DNA sequencing with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators

ABSTRACT

This invention provides a process for sequencing nucleic acids using 3′ modified deoxynucleotide analogues or 3′ modified deoxyinosine triphosphate analogues, and 3′ modified dideoxynucleotide analogues having a detectable marker attached thereto.

This application is a divisional of U.S. application Ser. No. 14/820,254, filed Aug. 6, 2015, now allowed, which is a continuation of U.S. application Ser. No. 12/734,229, filed Nov. 3, 2010, now U.S. Pat. No. 9,115,163, issued Aug. 25, 2015, which is a § 371 national stage of PCT International Application No. PCT/US2008/011913, filed Oct. 17, 2008, and claims the benefit of U.S. Provisional Applications Nos. 60/999,580, filed Oct. 19, 2007 and 60/999,575, filed Oct. 19, 2007, the contents of each of which are hereby incorporated by reference into this application.

This invention was made with government support under grant HG358205 awarded by the National Institutes of Health. The Government has certain rights in this invention.

Throughout this application, various publications are referenced in parentheses by number. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

The completion of the Human Genome Project (HGP) in early 2000 (1) was a monumental achievement with incredible amount of combined efforts among genome centers and scientists worldwide. The engine behind this decade long project was the Sanger sequencing method, which still currently maintains as the staple of large-scale genome sequencing methodology in high-throughput genome sequencing centers. The main reason behind this prolonged success was in the basic and efficient, yet elegant method that is Sanger dideoxy chain terminating reaction (2). With incremental improvements in this DNA sequencing technology including the use of laser induced fluorescent excitation of energy transfer dyes (3), engineered DNA polymerases (4) and capillary electrophoresis (5) as well as in the areas of sample preparation, informatics, and sequence analysis software (6-9), the Sanger sequencing platform has been able to maintain its status as champion in the sequencing world. Current state-of-the-art Sanger based DNA sequencers can produce over 700 bases of clearly readable sequence in a single run from templates up to 30 kb in length (10-12). However, as is with most of technological inventions, the continual improvements in this sequencing platform has come to a stagnant plateau, with the current cost estimate for producing a high-quality microbial genome draft sequence at around $10,000 per megabase pair. Current DNA sequencers based on the Sanger method allow up to 384 samples to be analyzed in parallel. However, one of the drawbacks to using electrophoresis for DNA separation is the deterioration of resolution due to band compressions. DNA sequences that are repeat rich and promote formation of secondary structures, such as hairpins, affect electrophoretic mobility, which also result in band compressions. This is the main reason behind maximum read-length limit for this sequencing method (13, 14). From a physics and engineering standpoint, the maximum read-length and parallelization based on capillary electrophoresis separation has already been reached (15).

At the onset of the post HGP-era, with realization of current sequencing platform's limitation, both public (National Human Genome Research Institute, NHGRI) and private genomic sciences sector (The J. Craig Venter Science Foundation and Archon X prize for genomics) have mandated a call for the development of next-generation sequencing technology that will reduce the cost of sequencing 100 and 10,000 fold in the next 5 to 10 years, respectively (16-19). With the development of a breakthrough DNA sequencing technology, which is already underway with heavy biotechnology industry involvement (20), it will allow for affordable genome sequencing. Genome research will be able to be conducted where it will be possible to move from studying single genes at a time to analyzing and comparing entire genomes. Recent data has demonstrated that the fundamental differences between many species including between mammals is not the overall number of genes, but lies at the more subtle regulatory level (21). This has led to the desire to sequence more genomes of closely related species as well as more human genomes. In addition, personalized medicine, gene expression analysis, splice form analysis and many other areas have demands for high-throughput sequencing projects that cannot be performed at the current speeds and costs. To overcome the limitations of the current sequencing technology based on electrophoresis using laser induced fluorescence detection (22-24), new methods must be developed which start from new paradigms to build a sequencer that can handle the new demands imposed by these new goals. Potential sequencing methods making significant steps forward into the new sequencing era include pyrosequencing (25-26), mass spectrometry sequencing (27-29), sequence specific detection of single-stranded DNA using engineered-nanopores (30), sequencing of single DNA molecules (31), polony sequencing (32) and sequencing by synthesis using cleavable fluorescent reversible terminators (33).

While fluorescent-based SBS methods have almost unlimited ability for parallelization, restricted only by the resolution of the imaging system, to date they have been limited to read lengths of about 35 bases. The successful implementation of sequencing by synthesis (SBS) is effectively dependent on the read length of the target DNA template. One of the major factors that determines the read length when performing SBS is the number of available templates. Our laboratory has recently developed two powerful approaches for SBS: 1) Hybrid SBS with nucleotide reversible terminator (NRTs, 3′-O—R₁-dNTPs) in combination with fluorescently labeled dideoxynucleotide (ddNTPs-R₂-fluorophore), and 2) SBS with cleavable fluorescent nucleotide reversible terminator (C-F-NRTs, 3′-O—R₁-dNTPs-R₂-fluorophore) (“Four-color DNA Sequencing with 3′-O-modified Nucleotide Reversible Terminators and Chemically Cleavable Fluorescent Dideoxynucleotides”. J. Guo, N. Xu, Z. Li, S. Zhang, J. Wu, D. Kim, M. S. Marma, Q. Meng, H. Cao, X. Li, S. Shi, L. Yu, S. Kalachikov, J. Russo, N. J. Turro, J. Ju. Proceedings of the National Academy of Sciences USA. 2008, 105, 9145-9150) (“Four-Color DNA Sequencing by Synthesis Using Cleavable Fluorescent Nucleotide Reversible Terminators”. J. Ju, D. Kim, L. Bi, Q. Meng, X. Bai, Z. Li, X. Li, M. S. Marma, S. Shi, J. Wu, J. R. Edwards, A. Romu, N. J. Turro. Proceedings of the National Academy of Sciences USA. 2006, 103, 19635-19640). Since the incorporation of ddNTPs-R₂-fluorophore into a strand of DNA permanently terminates further extensions of that template in the first approach and the incorporation and cleavage of C-F-NRTs leaves a tail of the modified nucleotide that causes possible steric hindrance to lower the incorporation efficiency of the subsequent base in the second approach, the total number of sequenceable templates decreases after each cycle of SBS reaction. Various means can be employed to minimize this rate of template reduction. Among those, a powerful method termed template “walking” can potentially diminish the negative effect of template termination or reduction and extend the read length of SBS at least two to three-fold.

SUMMARY OF THE INVENTION

A method for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:

-   -   a) contacting a plurality of the nucleic acids with (i) a         dideoxynucleotide triphosphate (ddNTP) analogue having the         structure:

-   -    wherein F is a fluorophore, L is a cleavable linker molecule         and the base is adenine, guanine, cytosine, uracil or thymine,         and wherein each base has a different fluorophore attached, and         wherein the fluorophore attached to each type of base differs in         its excitation or emission spectra from the fluorophores         attached to the other types of bases, (ii) a deoxynucleotide         triphosphate (dNTP) analogue having the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil, thymine or an inosine, and wherein R′ is a cleavable chemical group, (iii) a nucleic acid polymerase and (iv) at least two primers each of which hybridizes with a separate nucleic acid of the plurality of nucleic acids,

-   -    under conditions permitting a ddNTP analogue that is         complementary to the consecutive nucleotide residue to be         identified to form a phosphodiester bond with the 3′ end of one         of the primers and a dNTP analogue that is complementary to the         consecutive nucleotide residue to be identified to form a         phosphodiester bond with the 3′ end of another of the primers;     -   b) identifying the fluorophore of the ddNTP analogue which has         formed the phosphodiester bond thereby identifying the identify         of the consecutive nucleotide;     -   c) cleaving the fluorophore from the ddNTP analogue which has         formed the phosphodiester bond and cleaving the cleavable         chemical group from the dNTP which has formed the phosphodiester         bond;     -   d) iteratively repeating steps a) through c) for each of the         consecutive nucleotide residues to be identified until the final         consecutive nucleotide residue is to be identified;     -   e) repeating steps a) and b) to identify the final consecutive         nucleotide residue,         thereby determining the identity of each of the series of         consecutive nucleotide residues in the nucleic acid.

A method for determining the identity of consecutive nucleotide residues in a self-priming nucleic acid comprising:

-   -   a) contacting a plurality of the nucleic acids with (i) a         dideoxynucleotide triphosphate (ddNTP) analogue having the         structure:

-   -    wherein F is a fluorophore, L is a cleavable linker molecule         and the base is adenine, guanine, cytosine, uracil or thymine,         wherein each base has a different fluorophore attached, (ii) a         deoxynucleotide triphosphate (dNTP) analogue having the         structure:

wherein B is a base and is adenine, guanine, cytosine, uracil, thymine or an inosine, and wherein R′ is a cleavable chemical group, and (iii) a nucleic acid polymerase,

-   -    under conditions permitting a ddNTP analogue that is         complementary to the consecutive nucleotide residue to be         identified to form a phosphodiester bond with the 3′ end of one         of the self-priming nucleic acids and a dNTP analogue that is         complementary to the consecutive nucleotide residue to be         identified to form a phosphodiester bond with the 3′ end of         another of the self-priming nucleic acids;     -   b) identifying the fluorophore of the ddNTP analogue which has         formed the phosphodiester bond thereby identifying the identify         of the consecutive nucleotide;     -   c) cleaving the fluorophore from the ddNTP analogue which has         formed the phosphodiester bond and cleaving the cleavable         chemical group from the dNTP which has formed the phosphodiester         bond;     -   d) iteratively repeating steps a) through c) for each of the         consecutive nucleotide residues to be identified until the final         consecutive nucleotide residue is to be identified;     -   e) repeating steps a) and b) to identify the final consecutive         nucleotide residue,         thereby determining the identity of each of the series of         consecutive nucleotide residues in the nucleic acid.

A kit for sequencing a nucleic acid is provided comprising ddNTP analogues and dNTP analogues described herein and instructions for use in sequencing.

A compound having the structure:

wherein R′ is a cleavable chemical group.

A deoxyribonucleic acid having attached at a 3′ end thereof, by a phosphodiester bond, a compound having the structure:

wherein the O atom labeled α is the 3′ O atom of the deoxyribonucleic acid, the wavy line represents the remainder of the deoxyribonucleic acid that is 5′ relative to the 3′O, and wherein R′ is a cleavable chemical group.

A kit for sequencing a nucleic acid is provided comprising detectably-labeled dideoxynucleotide triphosphate analogues and the dITP analogue herein and instructions for use in sequencing.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Structures of the nucleotide reversible terminators, 3′-O—R₁-dATP, 3′-O—R₁-dCTP, 3′-O—R₁-dGTP, 3′-O—R₁-dTTP (left). Structures of the cleavable label modified dideoxynucleotide terminators, ddNTPs-R₂-Label (right), with 4 distinct labels corresponding to 4 different bases: ddCTP-R₂-Label 1, ddUTP-R₂-Label 2, ddATP-R₂-Label 3, and ddGTP-R₂-Label 4.

FIG. 2. Structures of ddNTPs-R₂-fluorophore, with the 4 fluorophores having distinct fluorescent emissions: ddCTP-R₂-Bodipy-FL-510 (λabs (max)=502 nm; λem (max)=510 nm), ddUTP-R₂-R6G (λabs (max)=525 nm; λem (max)=550 nm), ddATP-R₂-ROX (λabs (max)=575 nm; λem (max)=602 nm), and ddGTP-R₂-Bodipy-650 (λabs (max)=630 nm; λem (max)=650 nm).

FIG. 3. Reaction scheme of sequencing on a chip using combination of 3′-O-modified nucleotide reversible terminators (3′-O—R₁-dNTPs) and cleavable label modified dideoxynucleotide terminators (ddNTPs-R₂-label). In this sequencing approach, a chip is constructed with immobilized DNA templates that are able to self-prime for initiating the polymerase reaction. Four cleavable label modified dideoxynucleotides are designed such that each is attached with a unique label on the base through a cleavable linker (R₂). The four 3′-O-modified nucleotides have a small chemically reversible group (R₁) to cap the 3′-OH moiety. Upon adding the mixture of 3′-O—R₁-dNTPs and ddNTPs-R₂-label with the DNA polymerase, only the dideoxynucleotide/nucleotide analogue complementary to the next nucleotide on the template is incorporated by polymerase on each spot of the chip. The ratio of the two sets of nucleotides are tuned so that in each extension step, only a small amount of the labeled ddNTPs are incorporated into the self-priming DNA template to produce adequate signal for detection, while the rest are incorporated by the nucleotide reversible terminators. After removing the excess reagents and washing away any unincorporated dideoxynucleotide/nucleotide analogues, a 4 color fluorescence imager (if the label is a fluorescent dye) is used to image the surface of the chip, and the unique fluorescence emission from the specific fluorescent dye on the dideoxynucleotide analogues on each spot of the chip will yield the identity of the nucleotide. After imaging, the R₂-label and the R₁ protecting group will be removed by appropriate cleavage conditions to generate DNA products with the label removed and a free 3′-OH group with high yield, respectively. The self-primed DNA moiety on the chip at this stage is ready for the next cycle of the reaction to identify the next nucleotide sequence of the template DNA. The scheme may be performed mutatis mutandis for RNA, and may also be performed with a non-self-priming nucleic acid if primers are included in the reaction conditions.

FIG. 4. Exemplified structures of ddNTPs having photocleavably linked flurophores (ddNTPs-PC-fluorophore), with the 4 fluorophores having distinct fluorescent emissions: ddCTP-PC-Bodipy-FL-510 (λabs (max)=502 nm; λem (max)=510 nm), ddUTP-PC-R6G (λabs (max)=525 nm; λem (max)=550 nm), ddATP-PC-ROX (λabs (max)=575 nm; λem (max)=602 nm), and ddGTP-PC-Bodipy-650 (λabs (max)=630 nm; λem (max)=650 nm).

FIG. 5. A detailed scheme (top half of fig.) of polymerase reaction using all four photocleavable fluorescent dideoxynucleotides to extend with an “ddA”, “ddC”, “ddG” and “ddU” and the subsequent photocleavage reaction to cleave off the fluorophore from the DNA extension product. MALDI-TOF MS spectra (bottom half of fig.) verifying base specific incorporation of: (A) ddATP-PC-ROX (peak at 9,054 m/z) among pool of all four photocleavable fluorescent dideoxynucleotides, (B) the corresponding photocleavage product (8,315 m/z); (C) ddCTP-PC-Bodipy-FL-510 (peak at 8,788 m/z), (D) the corresponding photocleavage product (8,292 m/z); (E) ddGTP-PC-Bodipy-650 (peak at 9,043 m/z), (F) the corresponding photocleavage product (8,332 m/z); (G) ddUTP-PC-R6G (peak at 8,956 m/z) and (H) the corresponding photocleavage product (8,293 m/z).

FIG. 6. Structures of photocleavable dNTPs; exemplified are 3′-O-PC-dATP, 3′-O-PC-dCTP, 3′-O-PC-dGTP, and 3′-O-PC-dTTP.

FIG. 7. The polymerase extension scheme (left hand side of Fig.) and MALDI-TOF MS spectra of the four consecutive extension products and their photocleaved products (right hand side of Fig.). Primer extended with 3′-O-PC-dTTP (1), and its photocleaved product 2; Product 2 extended with 3′-O-PC-dGTP (3), and its photocleaved product 4; Product 4 extended with 3′-O-PC-dATP (5), and its photocleaved product 6; Product 6 extended with 3′-O-PC-dCTP (7), and its photocleaved product 8. After 10 seconds of irradiation at 355 nm the photocleavage is complete with the 3′-O-(2-nitrobenzyl) group cleaved from the extended DNA products.

FIG. 8. (A) Reaction scheme of Sanger/sequencing by synthesis hybrid sequencing on a chip using combination of photocleavable fluorescent dideoxynucleotides and 3′-O-PC-modified nucleotides. (B) The scanned 4-color fluorescence images (shown here in grayscale) for each step of Sanger/SBS hybrid sequencing on a chip: (1) incorporation of ddATP-PC-ROX and 3′-O-PC-dATP; (2) cleavage of PC-ROX and 3′-PC group; (3) incorporation of ddCTP-PC-Bodipy-FL-510 and 3′-O-PC-dCTP; (4) cleavage of PC-Bodipy-FL-510 and 3′-PC group; (5) incorporation of ddATP-PC-ROX and 3′-O-PC-dATP; (6) cleavage of PC-ROX and 3′-PC group; (7) incorporation of ddUTP-PC-R6G and 3′-O-PC-dTTP; (8) cleavage of PC-R6G and 3′-PC group; images (9) to (15) are similarly produced. (C) A plot (4-color sequencing data) of raw fluorescence emission intensity at the four designated emission wavelength of the four photocleavable fluorescent dideoxynucleotides.

FIG. 9. “Walking” Strategy 1

FIG. 10. Structures of the nucleotide reversible terminators

FIG. 11. Structures of cleavable fluorescent dideoxynucleotide terminators

FIG. 12. Hybrid SBS scheme

FIG. 13. Template “Walking” Method 1

FIG. 14. Template “Walking” Method 2

FIG. 15. Template “Walking” Method 3

FIG. 16. Template “Walking” Method 4

FIG. 17. Template “Walking” Method 5

FIG. 18. Structures of the nucleotide reversible terminators, 3′-O—N₃-dATP, 3′-O—N₃-dCTP, 3′-O—N₃-dGTP, 3′-O—N₃-dTTP

FIG. 19. Structures of cleavable fluorescent dideoxynucleotide terminators ddNTP-N₃-fluorophores, with the 4 fluorophores having distinct fluorescent emissions: ddCTP-N₃-Bodipy-FL-510 (λ_(abs (max))=502 nm; λ_(em (max))=510 nm), ddUTP-N₃-R6G (λ_(abs (max))=525 nm; λ_(em (max))=550 nm), ddATP-N₃-ROX (λ_(abs (max))=585 nm; λ_(em (max))=602 nm), and ddGTP-N₃-Cy5 (λ_(abs (max))=649 nm; λ_(em (max))=670 nm).

FIG. 20. (A) Staudinger reaction with TCEP to regenerate the 3′-OH group of the DNA extension product. (B) Staudinger reaction with TCEP to cleave the N₃-fluorophore from the dideoxynucleotide.

FIG. 21. Four-color DNA sequencing by the hybrid SBS approach

FIG. 22. Four-color DNA sequencing by the hybrid SBS after template “walking”

FIG. 23. General Scheme for SBS with C-F-NRTs

FIG. 24. Structure of 3′-O—N₃-dNTPs-N₃-fluorophore

FIG. 25. Four-color DNA SBS with 3′-O—N₃-dNTPs-N₃-fluorophore. (A) A SBS with C-F-NRTs scheme for four-color sequencing on a chip by using four 3′-O—N₃-dNTPs-N₃-fluorophore and 3′-O—N₃-dNTPs with ddNTPs capping. (B) Four-color fluorescence images for each step of the SBS: (1) incorporation of 3′-O—N₃-dCTP-N₃-Bodipy-Fl-510 and 3′-O—N₃-dCTP; (2) cleavage of N₃-Bodipy-Fl-510 and 3′-CH₂N₃ group; (3) incorporation of 3′-O—N₃-dATP-N₃-Rox and 3′-O—N₃-dATP; (4) cleavage of N₃-Rox and 3′-CH₂N₃ group; images 5-47 were produced similarly. (C) A plot (four-color sequencing data) of raw fluorescence emission intensity obtained by using 3′-O—N₃-dNTPs-N₃-fluorophore and 3′-O—N₃-dNTPs. The small groups of peaks between the identified bases are fluorescent background from the DNA chip.

FIG. 26. Template “Walking” Method 1 for SBS with C-F-NRTs

FIG. 27. Template “Walking” Method 2 for SBS with C-F-NRTs

FIG. 28. Template “Walking” Method 3 for SBS with C-F-NRTs

FIG. 29. Template “Walking” Method 4 for SBS with C-F-NRTs

FIG. 30. Template “Walking” Method 5 for SBS with C-F-NRTs

FIG. 31. “Walking” Strategy 2

FIG. 32. “Walking” Strategy 3

DETAILED DESCRIPTION OF THE INVENTION Terms

As used herein, and unless stated otherwise, each of the following terms shall have the definition set forth below.

A—Adenine;

C—Cytosine;

DNA—Deoxyribonucleic acid;

G—Guanine;

RNA—Ribonucleic acid;

T—Thymine; and

U—Uracil.

“Nucleic acid” shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J., USA).

“Type” of nucleotide refers to Adenine, Guanine, Cytosine, Thymine or Uracil. “Type” of base refers to adenine, uracil, cytosine, guanine or thymine.

“Mass tag” shall mean a molecular entity of a predetermined size which is capable of being attached by a cleavable bond to another entity.

“Solid substrate” shall mean any suitable medium present in the solid phase to which a nucleic acid or an agent may be affixed. Non-limiting examples include chips, beads and columns.

“Hybridize” shall mean the annealing of one single-stranded nucleic acid to another nucleic acid based on sequence complementarity. The propensity for hybridization between nucleic acids depends on the temperature and ionic strength of their milieu, the length of the nucleic acids and the degree of complementarity. The effect of these parameters on hybridization is well known in the art (see Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York.)

As used herein, hybridization of a primer sequence shall mean annealing sufficient such that the primer is extendable by creation of a phosphodiester bond.

As used herein, a ddNTP analogue, unless otherwise indicated, is a ddNTP substituted as its base with a linker molecule attached to a detectable marker, wherein the substitution is such that it does not prevent the ddNTP analogue from being incorporated by a nucleic acid polymerase into a primer extension stand resulting from a self-priming nucleic acid or from a primer hybridized to a nucleic acid of interest.

As used herein, a dNTP analogue, unless otherwise indicated, is a dNTP substituted as its base with a linker molecule attached to a detectable marker, wherein the substitution is such that it does not prevent the dNTP analogue from being incorporated by a nucleic acid polymerase into a primer extension stand resulting from a self-priming nucleic acid or from a primer hybridized to a nucleic acid of interest.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit (if appropriate) of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Embodiments of the Invention

A method is provided for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:

-   -   a) contacting a plurality of the nucleic acids with (i) a         dideoxynucleotide triphosphate (ddNTP) analogue having the         structure:

-   -    wherein F is a fluorophore, L is a cleavable linker molecule         and the base is adenine, guanine, cytosine, uracil or thymine,         and wherein each base has a different fluorophore attached, and         wherein the fluorophore attached to each type of base differs in         its excitation or emission spectra from the fluorophores         attached to the other types of bases, (ii) a deoxynucleotide         triphosphate (dNTP) analogue having the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil or thymine, and wherein R′ is a cleavable chemical group, (iii) a nucleic acid polymerase and (iv) at least two primers each of which hybridizes with a separate nucleic acid of the plurality of nucleic acids,

-   -    under conditions permitting a ddNTP analogue that is         complementary to the consecutive nucleotide residue to be         identified to form a phosphodiester bond with the 3′ end of one         of the primers and a dNTP analogue that is complementary to the         consecutive nucleotide residue to be identified to form a         phosphodiester bond with the 3′ end of another of the primers;     -   b) identifying the fluorophore of the ddNTP analogue which has         formed the phosphodiester bond thereby identifying the identify         of the consecutive nucleotide;     -   c) cleaving the fluorophore from the ddNTP analogue which has         formed the phosphodiester bond and cleaving the cleavable         chemical group from the dNTP which has formed the phosphodiester         bond;     -   d) iteratively repeating steps a) through c) for each of the         consecutive nucleotide residues to be identified until the final         consecutive nucleotide residue is to be identified;     -   e) repeating steps a) and b) to identify the final consecutive         nucleotide residue,     -   thereby determining the identity of each of the series of         consecutive nucleotide residues in the nucleic acid.

In an embodiment, the dNTP is a deoxyinosine triphosphate (dITP) analogue having the structure:

wherein R′ is a cleavable chemical group.

In an embodiment, the dNTP is adenine, guanine, cytosine, uracil or thymine. In an embodiment, the nucleic acid is DNA and the nucleic acid polymerase is a DNA polymerase. In anther embodiment, the nucleic acid is RNA and the polymerase is an RNA polymerase. In an embodiment, the plurality of nucleic acids are contacted in step a) with ddNTP analogues and dNTP analogues in a ddNTP:dNTP analogue ratio of about 1:10, 1:50, 1:250, or 1:500. In an embodiment, R′ is a nitrobenzyl group, an allyl group or a methylazido group. In an embodiment, the linker molecule is photocleavable. In an embodiment, the 1 carbon of the dideoxyribose is bonded to the 9 nitrogen of a guanine or adenine base or wherein the 1 carbon of the dideoxyribose is bonded to the 1 nitrogen of cytosine, thymine or uracil base. In an embodiment, the steps are performed in the order a), b), c), d) and e). In an embodiment, the steps are performed in the order a), c), b), d), and e).

In an embodiment, up to 1000 consecutive nucleotides are identified. In an embodiment, up to 1×10⁴ consecutive nucleotides are identified. In an embodiment, up to 1×10⁶ consecutive nucleotides are identified.

A method for determining the identity of consecutive nucleotide residues in a self-priming nucleic acid comprising:

-   -   a) contacting a plurality of the nucleic acids with (i) a         dideoxynucleotide triphosphate (ddNTP) analogue having the         structure:

-   -    wherein F is a fluorophore, L is a cleavable linker molecule         and the base is adenine, guanine, cytosine, uracil or thymine,         wherein each base has a different fluorophore attached, (ii) a         deoxynucleotide triphosphate (dNTP) analogue having the         structure:

wherein B is a base and is adenine, guanine, cytosine, uracil, thymine or an inosine, and wherein R′ is a cleavable chemical group, and (iii) a nucleic acid polymerase,

-   -    under conditions permitting a ddNTP analogue that is         complementary to the consecutive nucleotide residue to be         identified to form a phosphodiester bond with the 3′ end of one         of the self-priming nucleic acids and a dNTP analogue that is         complementary to the consecutive nucleotide residue to be         identified to form a phosphodiester bond with the 3′ end of         another of the self-priming nucleic acids;     -   b) identifying the fluorophore of the ddNTP analogue which has         formed the phosphodiester bond thereby identifying the identify         of the consecutive nucleotide;     -   c) cleaving the fluorophore from the ddNTP analogue which has         formed the phosphodiester bond and cleaving the cleavable         chemical group from the dNTP which has formed the phosphodiester         bond;     -   d) iteratively repeating steps a) through c) for each of the         consecutive nucleotide residues to be identified until the final         consecutive nucleotide residue is to be identified;     -   e) repeating steps a) and b) to identify the final consecutive         nucleotide residue,     -   thereby determining the identity of each of the series of         consecutive nucleotide residues in the nucleic acid.

In an embodiment, the dNTP is a deoxyinosine triphosphate (dITP) analogue having the structure:

wherein R′ is a cleavable chemical group.

In an embodiment, the dNTP is adenine, guanine, cytosine, uracil or thymine. In an embodiment, nucleic acid is DNA and the nucleic acid polymerase is a DNA polymerase. In another embodiment, the nucleic acid is RNA and the polymerase is an RNA polymerase. In an embodiment, the plurality of nucleic acids are contacted in step a) with ddNTP analogues and dNTP analogues in a ddNTP:dNTP analogue ratio of about 1:10, 1:50, 1:250, or 1:500. In an embodiment, R′ is a nitrobenzyl group, an allyl group or a methylazido group. In an embodiment, the linker molecule is photocleavable. In an embodiment, the 1 carbon of the dideoxyribose is bonded to the 9 nitrogen of a guanine or adenine base or the 1 carbon of the dideoxyribose is bonded to the 1 nitrogen of cytosine, thymine or uracil base. In an embodiment, the steps are performed in the order a), b), c), d) and e). In an embodiment, the steps are performed in the order a), c), b), d), and e).

In an embodiment, up to 1000 consecutive nucleotides are identified. In an embodiment, up to 1×10⁴ consecutive nucleotides are identified. In an embodiment, up to 1×10⁶ consecutive nucleotides are identified.

A kit for sequencing a nucleic acid is provided comprising ddNTP analogues and dNTP analogues described herein and instructions for use in sequencing.

A compound is provided having the structure:

wherein R′ is a cleavable chemical group.

In an embodiment, R′ is a nitrobenzyl group, an allyl group or a methylazido group. In an embodiment, the base has a detectable marker cleavably linked thereto.

A deoxyribonucleic acid is provided having attached at a 3′ end thereof, by a phosphodiester bond, a compound having the structure:

wherein the O atom labeled α is the 3′ O atom of the deoxyribonucleic acid, the wavy line represents the remainder of the deoxyribonucleic acid that is 5′ relative to the 3′O, and wherein R′ is a cleavable chemical group.

In an embodiment, R′ is a nitrobenzyl group, an allyl group or a methylazido group. In an embodiment, the deoxyribonucleic acid is attached to a solid surface.

R′ can have the structure:

where the wavy line represents the point of attachment to the 3′ O atom.

L, as the linker molecule, can comprise or consist of the structure:

wherein the left hand wavy line represents the point of attachment to a base, or further molecule connecting to the base, and the right hand wavy line represents the point of attachment to a fluorophore or other detectable marker, or a further molecule connecting to the fluorophore or other detectable marker.

A kit for sequencing a nucleic acid is provided comprising ddNTP analogues and dITP analogues described herein and instructions for use in sequencing.

In the methods, compositions and kits disclosed herein, the dNTP:ddNTP ratio can be altered to optimize results. Embodiments include 10:1, 100:1, 1000:1, 10,000:1, 100,000:1, 10-100:1, 100-1,000:1, 1,000-10,1000:1, 10,000-100,000:1 of dNTP analogues to ddNTP analogues.

This invention provides the instant method, wherein the detectable bound to the base via a cleavable linker is a dye, a fluorophore, a chromophore, a combinatorial fluorescence energy transfer tag, a mass tag, or an electrophore.

This invention also provides the instant method, wherein the primer is a self-priming moiety.

This invention also provides the instant method, wherein the DNA is bound to a solid substrate. This invention also provides the instant method, wherein the DNA is bound to the solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry. This invention also provides the instant method, wherein the DNA is bound to the solid substrate via a polyethylene glycol molecule. This invention also provides the instant method, wherein the DNA is alkyne-labeled. This invention also provides the instant method, wherein the DNA is bound to the solid substrate via a polyethylene glycol molecule and the solid substrate is azide-functionalized. This invention also provides the instant method, wherein the DNA is immobilized on the solid substrate via an azido linkage, an alkynyl linkage, or biotin-streptavidin interaction. Immobilization of nucleic acids is described in Immobilization of DNA on Chips II, edited by Christine Wittmann (2005), Springer Verlag, Berlin, which is hereby incorporated by reference.

This invention also provides the instant method, wherein the solid substrate is in the form of a chip, a bead, a well, a capillary tube, a slide, a wafer, a filter, a fiber, a porous media, or a column. This invention also provides the instant method, wherein the solid substrate is gold, quartz, silica, plastic, glass, diamond, silver, metal, or polypropylene. This invention also provides the instant method, wherein the solid substrate is porous. Chips or beads may be made from materials common for DNA microarrays, for example glass or nylon. Beads/micro-beads may be in turn immobilized to chips.

This invention also provides the instant method, wherein about 1000 or fewer copies of the DNA are bound to the solid substrate. This invention also provides the instant invention wherein 2×10⁷, 1×10⁷, 1×10⁶ or 1×10⁴ or fewer copies of the DNA are bound to the solid substrate.

This invention also provides the instant method, wherein the nucleotide analogues comprise one of the fluorophores Cy5, Bodipy-FL-510, ROX and R6G.

This invention also provides the instant method, wherein the four deoxynucleotide analogues are 3′-O-allyl-dGTP, 3′-O-allyl-dCTP, 3′-O-allyl-dATP and 3′-O-allyl-dUTP. This invention also provides the instant method, wherein the four deoxynucleotide analogues are 3′-O-methylazido-dGTP, 3′-O-methylazido-dCTP, 3′-O-methylazido-dATP and 3′-O-methylazido-dUTP. It is understood that in other embodiments the deoxynucleotide or dideoxynucleotide analogues are photocleavable. For example, photocleavable linkers such as 2-nitrobenzyl can replace any of the allyl moieties in the analogues described herein. For example, 3′-O-2-nitrobenzyl-dGTP, 3′-O-2-nitrobenzyl-dATP, 3′-O-2-nitrobenzyl-dGTP, 3′-O-2-nitrobenzyl-dATP. One of skill in the art would recognize various other chemically cleavable or photochemically cleavable moieties or linkers that can be used in place of the examples described herein. Additionally, the unique labels may also be varied, and the examples set forth herein are non-limiting. In an embodiment UV light is used to photochemically cleave the photochemically cleavable linkers and moieties.

This invention also provides the instant method, wherein the deoxynucleotide analogue is 3′-O-allyl-dITP, or 3′-O-methylazido-dITP. It is understood that in other embodiments the modifications of the deoxynucleotide or dideoxynucleotide analogues are photocleavable. For example, photocleavable linkers, such as 2-nitrobenzyl, can replace any of the allyl moieties in the analogues described herein. For example, 3′-O-2-nitrobenzyl-dITP. One of skill in the art would recognize various other chemically cleavable or photochemically cleavable moieties or linkers that can be used in place of the examples described herein. Additionally, the unique labels may also be varied, and the examples set forth herein are non-limiting. In an embodiment UV light is used to photochemically cleave the photochemically cleavable linkers and moieties.

This invention also provides the instant method, wherein the dideoxynucleotide terminators are have a detectable marker, for example a fluorophore, a mass tag, a chromophore etc., attached to the base thereof via an allyl linker, a photocleavable linker or other linkers known in the art including the photocleavable linkers set forth herein.

This invention also provides the instant method, wherein the dideoxynucleotide terminators are 3′-O-allyl-dGTP, 3′-O-allyl-dCTP, 3′-O-allyl-dATP and 3′-O-allyl-dUTP, 3′-O-2-nitrobenzyl-dGTP, 3′-O-2-nitrobenzyl-dCTP, 3′-O-2-nitrobenzyl-dATP and 3′-O-2-nitrobenzyl-dUTP, 3-O-methylazido-dGTP, 3′-O-methylazido-dCTP, 3′-O-methylazido-dATP and 3′-Omethylazido-dUTP, 3′-O-2-methylazido-dGTP, 3′-O-2-methylazido-dCTP, 3′-O-2-methylazido-dATP and 3′-O-2-methylazido-dUTP.

This invention also provides the instant method, wherein the DNA polymerase is a 9° N polymerase or a variant thereof. DNA polymerases which can be used in the instant invention include, for example E. coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase™, Taq DNA polymerase and 9° N polymerase (exo-) A485L/Y409V.RNA polymerases which can be used in the instant invention include, for example, Bacteriophage SP6, T7 and T3 RNA polymerases.

This invention also provides the instant method, wherein the DNA or nucleic acid being sequenced (i.e. consecutive nucleotides thereof being identified) is bound to the solid substrate via a polyethylene glycol molecule and the solid substrate is azide-functionalized or the DNA is immobilized on the solid substrate via an azido linkage, an alkynyl linkage, or biotin-streptavidin interaction. Immobilization of nucleic acids is described in Immobilization of DNA on Chips II, edited by Christine Wittmann (2005), Springer Verlag, Berlin, which is hereby incorporated by reference.

Methods for production of cleavably capped and/or cleavably linked nucleotide analogues are disclosed in U.S. Pat. No. 6,664,079, which is hereby incorporated by reference. Combinatorial fluorescence energy tags and methods for production thereof are disclosed in U.S. Pat. No. 6,627,748, which is hereby incorporated by reference.

In an embodiment, the DNA or nucleic acid is attached/bound to the solid surface by covalent site-specific coupling chemistry compatible with DNA.

A method for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:

-   -   a) contacting a plurality of the nucleic acids with (i) a         dideoxynucleotide triphosphate (ddNTP) analogue having the         structure:

-   -   wherein F is a fluorophore, L is a cleavable linker molecule and         the base is adenine, guanine, cytosine, uracil or thymine, and         wherein each base has a different fluorophore attached, and         wherein the fluorophore attached to each type of base differs in         its excitation or emission spectra from the fluorophores         attached to the other types of bases, (ii) a deoxynucleotide         triphosphate (dNTP) analogue having the structure:

-   -   wherein B is a base and is adenine, guanine, cytosine, uracil,         thymine or an inosine, and wherein R′ is a cleavable chemical         group, (iii) a nucleic acid polymerase and (iv) at least two         primers each of which hybridizes with a separate nucleic acid of         the plurality of nucleic acids,     -   under conditions permitting a ddNTP analogue that is         complementary to the consecutive nucleotide residue to be         identified to form a phosphodiester bond with the 3′ end of one         of the primers and a dNTP analogue that is complementary to the         consecutive nucleotide residue to be identified to form a         phosphodiester bond with the 3′ end of another of the primers;     -   b) identifying the fluorophore of the ddNTP analogue which has         formed the phosphodiester bond thereby identifying the identify         of the consecutive nucleotide;     -   c) cleaving the fluorophore from the ddNTP analogue which has         formed the phosphodiester bond and cleaving the cleavable         chemical group from the dNTP which has formed the phosphodiester         bond;     -   d) iteratively repeating steps a) through c) for each of the         consecutive nucleotide residues to be identified until the final         consecutive nucleotide residue is to be identified;     -   e) repeating steps a) and b) to identify the final consecutive         nucleotide residue,     -   thereby determining the identity of each of the series of         consecutive nucleotide residues in the nucleic acid.     -   In an embodiment the dNTP is a deoxyinosine triphosphate (dITP)         analogue having the structure:

wherein R′ is a cleavable chemical group.

-   -   In an embodiment the base of the dNTP is adenine, guanine,         cytosine, uracil or thymine.     -   In an embodiment the nucleic acid is DNA and the nucleic acid         polymerase is a DNA polymerase.     -   In an embodiment wherein the plurality of nucleic acids are         contacted in step a) with ddNTP analogues and dNTP analogues in         a ddNTP analogues:dNTP analogues ratio of about 1:10, 1:50,         1:250, or 1:500.     -   In an embodiment R′ is a nitrobenzyl group, an allyl group or a         methylazido group.     -   In an embodiment the linker molecule is photocleavable.     -   In an embodiment the 1 carbon of the dideoxyribose is bonded to         the 9 nitrogen of a guanine or adenine base or wherein the 1         carbon of the dideoxyribose is bonded to the 1 nitrogen of         cytosine, thymine or uracil base.     -   In an embodiment the 1 carbon of the dideoxyribose is bonded to         the 9 nitrogen of an inosine base.     -   In an embodiment up to 1000 consecutive nucleotides are         identified.     -   In an embodiment up to 1×10⁴ consecutive nucleotides are         identified.     -   In an embodiment up to 1×10⁶ consecutive nucleotides are         identified.     -   A method for determining the identity of consecutive nucleotide         residues in a self-priming nucleic acid comprising:         -   a) contacting a plurality of the nucleic acids with (i) a             dideoxynucleotide triphosphate (ddNTP) analogue having the             structure:

-   -   -   wherein F is a fluorophore, L is a cleavable linker molecule             and the base is adenine, guanine, cytosine, uracil or             thymine, wherein each base has a different fluorophore             attached, (ii) a deoxynucleotide triphosphate (dNTP)             analogue having the structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine,             uracil, thymine or an inosine, and wherein R′ is a cleavable             chemical group, and (iii) a nucleic acid polymerase,         -   under conditions permitting a ddNTP analogue that is             complementary to the consecutive nucleotide residue to be             identified to form a phosphodiester bond with the 3′ end of             one of the self-priming nucleic acids and a dNTP analogue             that is complementary to the consecutive nucleotide residue             to be identified to form a phosphodiester bond with the 3′             end of another of the self-priming nucleic acids;         -   b) identifying the fluorophore of the ddNTP analogue which             has formed the phosphodiester bond thereby identifying the             identify of the consecutive nucleotide;         -   c) cleaving the fluorophore from the ddNTP analogue which             has formed the phosphodiester bond and cleaving the             cleavable chemical group from the dNTP which has formed the             phosphodiester bond;         -   d) iteratively repeating steps a) through c) for each of the             consecutive nucleotide residues to be identified until the             final consecutive nucleotide residue is to be identified;         -   e) repeating steps a) and b) to identify the final             consecutive nucleotide residue,

    -    thereby determining the identity of each of the series of         consecutive nucleotide residues in the nucleic acid.

    -   In an embodiment the dNTP is a deoxyinosine triphosphate (dITP)         analogue having the structure:

wherein R′ is a cleavable chemical group.

-   -   In an embodiment the base of the dNTP is adenine, guanine,         cytosine, uracil or thymine.     -   In an embodiment the nucleic acid is DNA and the nucleic acid         polymerase is a DNA polymerase.     -   In an embodiment the plurality of nucleic acids are contacted in         step a) with ddNTP analogues and dNTP analogues in a ddNTP         analogues:dNTP analogue ratio of about 1:10, 1:50, 1:250, or         1:500.     -   In an embodiment R′ is a nitrobenzyl group, an allyl group or a         methylazido group.     -   In an embodiment the linker molecule is photocleavable.     -   In an embodiment the 1 carbon of the dideoxyribose is bonded to         the 9 nitrogen of a guanine or adenine base or wherein the 1         carbon of the dideoxyribose is bonded to the 1 nitrogen of         cytosine, thymine or uracil base.     -   In an embodiment the 1 carbon of the dideoxyribose is bonded to         the 9 nitrogen of an inosine base.     -   In an embodiment up to 1000 consecutive nucleotides are         identified.     -   In an embodiment up to 1×10⁴ consecutive nucleotides are         identified.     -   In an embodiment up to 1×10⁶ consecutive nucleotides are         identified.     -   In an embodiment the steps are performed in the order a), b),         c), d), and e).     -   In an embodiment the steps are performed in the order a), c),         b), d), and e).     -   A kit for sequencing a nucleic acid is provided comprising ddNTP         analogues and dNTP analogues described herein and instructions         for use in sequencing.     -   A compound having the structure:

wherein R′ is a cleavable chemical group.

-   -   In an embodiment R′ is a nitrobenzyl group, an allyl group or a         methylazido group.     -   In an embodiment the base has a detectable marker cleavably         linked thereto.     -   A deoxyribonucleic acid having attached at a 3′ end thereof, by         a phosphodiester bond, a compound having the structure:

wherein the O atom labeled α is the 3′ O atom of the deoxyribonucleic acid, the wavy line represents the remainder of the deoxyribonucleic acid that is 5′ relative to the 3′O, and wherein R′ is a cleavable chemical group.

-   -   In an embodiment R′ is a nitrobenzyl group, an allyl group or a         methylazido group.     -   In an embodiment the deoxyribonucleic acid is attached to a         solid surface.     -   A kit for sequencing a nucleic acid is provided comprising         detectably-labeled dideoxynucleotide triphosphate analogues and         the dITP analogue of claim 31 and instructions for use in         sequencing.     -   A method for determining the identity of each of a series of         consecutive nucleotide residues in a nucleic acid comprising:         -   a) contacting a plurality of the nucleic acids with (i) at             least four different dideoxynucleotide triphosphate (ddNTP)             analogues, each having the structure:

-   -   -   wherein F is a fluorophore, b is a base which is adenine,             guanine, cytosine, uracil or thymine, wherein the             fluorophore attached through a linker to each type of base             differs in its emission or excitation spectra from a             fluorophore attached to each of the remaining types of             bases, and each of the four ddNTP analogues differs from the             remaining three ddNTP analogues by having a different base,             wherein L is a cleavable linker molecule, (ii) at least four             deoxynucleotide triphosphate (dNTP) analogues having the             structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine,             uracil, or thymine, and wherein R′ is a cleavable chemical             group, wherein each of the four dNTP analogues differs from             the remaining three dNTP analogues by having a different             base, (iii) a nucleic acid polymerase and (iv) a plurality             of nucleic acid primers which can each hybridize with a             separate one of each of the plurality of nucleic acids,             under conditions permitting (a) one of the four ddNTP             analogues that is complementary to the consecutive             nucleotide residue to be identified to form a phosphodiester             bond with the 3′ end of one of the nucleic acid primers and             thereby extend the primer and (b) one of the four dNTP             analogues that is complementary to a consecutive nucleotide             residue to be identified to form a phosphodiester bond with             the 3′ end of another one of the nucleic acid primers and             thereby extend that primer;         -   b) identifying the fluorophore of the ddNTP analogue which             has formed the phosphodiester bond, thereby identifying the             consecutive nucleotide;         -   c) cleaving the linker attaching the fluorophore of the             ddNTP analogue which has formed the phosphodiester bond and             cleaving the cleavable chemical group from the dNTP which             has formed the phosphodiester bond;         -   d) iteratively repeating steps a) through c) for each of the             consecutive nucleotide residues to be identified until the             final consecutive nucleotide residue is to be identified;         -   e) repeating steps a) and b) to identify the final             consecutive nucleotide residue,         -   f) denaturing the extended primers so that they de-hybridize             from the plurality of nucleic acids;         -   g) contacting the plurality of nucleic acids with (i) at             least four different deoxynucleotide triphosphate (dNTP)             analogues each comprising a base chosen from adenine,             thymine, cytosine, uracil, inosine, or 5-nitroindole, each             differing from a deoxynucleotide triphosphate by having a             cleavable chemical group attached to the 3′ O-atom of the             dNTP, (ii) a nucleic acid polymerase and (iii) a plurality             of second nucleic acid primers which each separately             hybridize with a separate one of the plurality of nucleic             acids, under conditions permitting one of the four dNTP             analogues that is complementary to the consecutive             nucleotide residue to be identified to form a phosphodiester             bond with the 3′ end of one of the second nucleic acid             primers and thereby extend that second primer;         -   h) cleaving the chemical group from the 3′ O-atom of the             dNTP analogue which has formed the phosphodiester bond so as             to thereby permit incorporation of a further dNTP analogue             into the extended second nucleic acid primer;         -   i) iteratively repeating steps g) and h) until the second             primer is extended up to and including a residue             corresponding to the final consecutive nucleotide residue             identified in step e);         -   j) contacting the plurality of extended second primers             with (i) at least four different dideoxynucleotide             triphosphate (ddNTP) analogues, each having the structure:

-   -   -   wherein F is a fluorophore, b is a base which is adenine,             guanine, cytosine, uracil or thymine, wherein the             fluorophore attached through a linker to each type of base             differs in its emission or excitation spectra from a             fluorophore attached to each of the remaining types of             bases, and each of the four ddNTP analogues differs from the             remaining three ddNTP analogues by having a different base,             wherein L is a cleavable linker molecule, (ii) at least four             deoxynucleotide triphosphate (dNTP) analogues having the             structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine,             uracil, or thymine, and wherein R′ is a cleavable chemical             group, wherein each of the four dNTP analogues differs from             the remaining three dNTP analogues by having a different             base, and (iii) a nucleic acid polymerase, under conditions             permitting (a) one of the four ddNTP analogues that is             complementary to the next consecutive nucleotide residue to             be identified to form a phosphodiester bond with the 3′ end             of one of the extended second nucleic acid primers and             thereby extend the second primer and (b) one of the four             dNTP analogues that is complementary to a consecutive             nucleotide residue to be identified to form a phosphodiester             bond with the 3′ end of another one of the extended second             nucleic acid primers and thereby extend that second primer;         -   k) identifying the fluorophore of the ddNTP analogue which             has formed the phosphodiester bond, thereby identifying the             consecutive nucleotide;         -   l) cleaving the linker attaching the fluorophore of the             ddNTP analogue which has formed the phosphodiester bond and             cleaving the cleavable chemical group from the dNTP which             has formed the phosphodiester bond;         -   m) iteratively repeating steps j) through l) for each of the             consecutive nucleotide residues to be identified until the             final consecutive nucleotide residue is to be identified;         -   n) repeating steps j) and k) to identify the final             consecutive nucleotide residue, so as to thereby determine             the identity of each of the series of consecutive nucleotide             residues in the nucleic acid.

    -   In an embodiment the linker in each of step a) and j)         independently each comprise the structure:

or the structure:

wherein α represents a point of attachment to the base and β represents a point of attachment to the fluorophore, and wherein R is a cleavable chemical group.

-   -   In an embodiment a linker is cleaved by contacting the linker         with tris(2-carboxyethyl)phosphine.     -   In an embodiment one or more linkers are photocleavable or         chemically cleavable.     -   In an embodiment one or more chemical groups are photocleavable         or chemically cleavable.     -   In an embodiment R in the structures set forth in steps a) and         or j) is independently chosen from a —N₃ group or an allyl         group.     -   In an embodiment the cleavable chemical group in step g) is         independently chosen from a —N₃ group or an allyl group.     -   A method for determining the identity of each of a series of         consecutive nucleotide residues in a nucleic acid comprising:         -   a) contacting a plurality of the nucleic acids with (i) at             least four different dideoxynucleotide triphosphate (ddNTP)             analogues, each having the structure:

-   -   -   wherein F is a fluorophore, b is a base which is adenine,             guanine, cytosine, uracil or thymine, wherein the             fluorophore attached through a linker to each type of base             differs in its emission or excitation spectra from a             fluorophore attached to each of the remaining types of             bases, and each of the four ddNTP analogues differs from the             remaining three ddNTP analogues by having a different base,             wherein L is a cleavable linker molecule, (ii) at least four             deoxynucleotide triphosphate (dNTP) analogues having the             structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine,             uracil, or a thymine, and wherein R′ is a cleavable chemical             group, wherein each of the four dNTP analogues differs from             the remaining three dNTP analogues by having a different             base, (iii) a nucleic acid polymerase and (iv) a plurality             of nucleic acid primers which can each hybridize with a             separate one of each of the plurality of nucleic acids,             under conditions permitting (a) one of the four ddNTP             analogues that is complementary to the consecutive             nucleotide residue to be identified to form a phosphodiester             bond with the 3′ end of one of the nucleic acid primers and             thereby extend the primer and (b) one of the four dNTP             analogues that is complementary to a consecutive nucleotide             residue to be identified to form a phosphodiester bond with             the 3′ end of another one of the nucleic acid primers and             thereby extend that primer;         -   b) identifying the fluorophore of the ddNTP analogue which             has formed the phosphodiester bond, thereby identifying the             consecutive nucleotide;         -   c) cleaving the linker attaching the fluorophore of the             ddNTP analogue which has formed the phosphodiester bond and             cleaving the cleavable chemical group from the dNTP which             has formed the phosphodiester bond;         -   d) iteratively repeating steps a) through c) for each of the             consecutive nucleotide residues to be identified until the             final consecutive nucleotide residue is to be identified;         -   e) repeating steps a) and b) to identify the final             consecutive nucleotide residue,         -   f) denaturing the extended primers so as to de-hybridize             them from the plurality of nucleic acids;         -   g) contacting the nucleic acids with (i) three different             types of deoxynucleotide triphosphate, (ii) a nucleic acid             polymerase and (iii) a second plurality of nucleic acid             primers which each hybridize with a separate one of the             plurality of nucleic acids, under conditions permitting one             of the three dNTP analogues that is complementary to the             consecutive nucleotide residue to be identified to form a             phosphodiester bond with the 3′ end of the second nucleic             acid primer and thereby extend the second nucleic acid             primer;         -   h) contacting the nucleic acid with (i) three different             types of deoxynucleotide triphosphate, wherein at least one             of the types of deoxynucleotide triphosphate is not used in             step g), under conditions permitting one of the three dNTP             that is complementary to the consecutive nucleotide residue             to be identified to form a phosphodiester bond with the 3′             end of the extended second nucleic acid primer and thereby             further extend the second nucleic acid primer;         -   i) repeating steps g) and h) until the second nucleic acid             primer is extended up to and including a residue             corresponding to the final consecutive nucleotide residue             identified in step e)         -   j) contacting the plurality of extended second primers             with (i) at least four different dideoxynucleotide             triphosphate (ddNTP) analogues, each having the structure:

-   -   -   wherein F is a fluorophore, b is a base which is adenine,             guanine, cytosine, uracil or thymine, wherein the             fluorophore attached through a linker to each type of base             differs in its emission or excitation spectra from a             fluorophore attached to each of the remaining types of             bases, and each of the four ddNTP analogues differs from the             remaining three ddNTP analogues by having a different base,             wherein L is a cleavable linker molecule, (ii) at least four             deoxynucleotide triphosphate (dNTP) analogues having the             structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine,             uracil, or a thymine, and wherein R′ is a cleavable chemical             group, wherein each of the four dNTP analogues differs from             the remaining three dNTP analogues by having a different             base, and (iii) a nucleic acid polymerase,         -   under conditions permitting (a) one of the four ddNTP             analogues that is complementary to the consecutive             nucleotide residue to be identified to form a phosphodiester             bond with the 3′ end of one of the extended second nucleic             acid primers and thereby extend the second primer and (b)             one of the four dNTP analogues that is complementary to a             consecutive nucleotide residue to be identified to form a             phosphodiester bond with the 3′ end of another one of the             extended second nucleic acid primers and thereby extend that             second primer;         -   k) identifying the fluorophore of the ddNTP analogue which             has formed the phosphodiester bond, thereby identifying the             consecutive nucleotide;         -   l) cleaving the linker attaching the fluorophore of the             ddNTP analogue which has formed the phosphodiester bond and             cleaving the cleavable chemical group from the dNTP which             has formed the phosphodiester bond;         -   m) iteratively repeating steps j) through 1) for each of the             consecutive nucleotide residues to be identified until the             final consecutive nucleotide residue is to be identified;         -   n) repeating steps j) and k) to identify the final             consecutive nucleotide residue, so as to thereby determine             the identity of each of the series of consecutive nucleotide             residues in the nucleic acid.

    -   In an embodiment in steps g) and h) the three types of dNTPs are         chosen from the group dATP, dCTP, dGTP and dTTP.

    -   In an embodiment the linker in each of step a) and j)         independently each comprise the structure:

or the structure:

wherein α represents a point of attachment to the base and β represents a point of attachment to the fluorophore, and wherein R is a cleavable chemical group.

-   -   In an embodiment a linker is cleaved by contacting the linker         with tris(2-carboxyethyl)phosphine.     -   In an embodiment one or more linkers are photocleavable or         chemically cleavable.     -   In an embodiment one or more chemical groups are photocleavable         or chemically cleavable.     -   In an embodiment R in the structures set forth in steps a) and         or j) is independently chosen from a —N₃ group or an allyl         group.     -   In an embodiment the cleavable chemical group in step g) is         independently chosen from a —N₃ group or an allyl group.     -   A method for determining the identity of each of a series of         consecutive nucleotide residues in a nucleic acid comprising:         -   a) contacting a plurality of the nucleic acids with (i) at             least four different dideoxynucleotide triphosphate (ddNTP)             analogues, each having the structure:

-   -   -   wherein F is a fluorophore, b is a base which is adenine,             guanine, cytosine, uracil or thymine, wherein the             fluorophore attached through a linker to each type of base             differs in its emission or excitation spectra from a             fluorophore attached to each of the remaining types of             bases, and each of the four ddNTP analogues differs from the             remaining three ddNTP analogues by having a different base,             wherein L is a cleavable linker molecule, (ii) at least four             deoxynucleotide triphosphate (dNTP) analogues having the             structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine,             uracil, or a thymine, and wherein R′ is a cleavable chemical             group, wherein each of the four dNTP analogues differs from             the remaining three dNTP analogues by having a different             base, (iii) a nucleic acid polymerase and (iv) a plurality             of nucleic acid primers which can each hybridize with a             separate one of each of the plurality of nucleic acids,         -   under conditions permitting (a) one of the four ddNTP             analogues that is complementary to the consecutive             nucleotide residue to be identified to form a phosphodiester             bond with the 3′ end of one of the nucleic acid primers and             thereby extend the primer and (b) one of the four dNTP             analogues that is complementary to a consecutive nucleotide             residue to be identified to form a phosphodiester bond with             the 3′ end of another one of the nucleic acid primers and             thereby extend that primer;         -   b) identifying the fluorophore of the ddNTP analogue which             has formed the phosphodiester bond, thereby identifying the             consecutive nucleotide;         -   c) cleaving the linker attaching the fluorophore of the             ddNTP analogue which has formed the phosphodiester bond and             cleaving the cleavable chemical group from the dNTP which             has formed the phosphodiester bond;         -   d) iteratively repeating steps a) through c) for each of the             consecutive nucleotide residues to be identified until the             final consecutive nucleotide residue is to be identified;         -   e) repeating steps a) and b) to identify the final             consecutive nucleotide residue,         -   f) denaturing the extended primers so as to de-hybridize             them from the plurality of nucleic acids;         -   g) contacting the nucleic acid with (i) three different             types of deoxynucleotide triphosphates, (ii) a             deoxynucleotide triphosphate analogue, differing from a             deoxynucleotide triphosphate by having a cleavable chemical             group attached to the 3′ O-atom of the dNTP analogue and             differing from the three different types of deoxynucleotide             triphosphates by having a different base therefrom, (iii) a             nucleic acid polymerase and (iv) a second nucleic acid             primer which hybridizes with the nucleic acid, under             conditions permitting one of the three dNTPs or the dNTP             analogue that is complementary to the consecutive nucleotide             residue to be identified to form a phosphodiester bond with             the 3′ end of one of the second nucleic acid primers and             thereby extend that second nucleic acid primer;         -   h) cleaving the cleavable chemical group from the 3′-O-atom             group;         -   i) repeating steps g) and h) until the second nucleic acid             primer is extended up to and including a residue             corresponding to the final consecutive nucleotide residue             identified in step e)         -   j) contacting the plurality of extended second primers             with (i) at least four different dideoxynucleotide             triphosphate (ddNTP) analogues, each having the structure:

-   -   -   wherein F is a fluorophore, b is a base which is adenine,             guanine, cytosine, uracil or thymine, wherein the             fluorophore attached through a linker to each type of base             differs in its emission or excitation spectra from a             fluorophore attached to each of the remaining types of             bases, and each of the four ddNTP analogues differs from the             remaining three ddNTP analogues by having a different base,             wherein L is a cleavable linker molecule, (ii) at least four             deoxynucleotide triphosphate (dNTP) analogues having the             structure:

-   -   -   wherein B is a base and is adenine, guanine, cytosine,             uracil, or thymine, and wherein R′ is a cleavable chemical             group, wherein each of the four dNTP analogues differs from             the remaining three dNTP analogues by having a different             base, and (iii) a nucleic acid polymerase,         -   under conditions permitting (a) one of the four ddNTP             analogues that is complementary to the consecutive             nucleotide residue to be identified to form a phosphodiester             bond with the 3′ end of one of the extended second nucleic             acid primers and thereby extend the second primer and (b)             one of the four dNTP analogues that is complementary to a             consecutive nucleotide residue to be identified to form a             phosphodiester bond with the 3′ end of another one of the             extended second nucleic acid primers and thereby extend that             second primer;         -   k) identifying the fluorophore of the ddNTP analogue which             has formed the phosphodiester bond, thereby identifying the             consecutive nucleotide;         -   l) cleaving the linker attaching the fluorophore of the             ddNTP analogue which has formed the phosphodiester bond and             cleaving the cleavable chemical group from the dNTP which             has formed the phosphodiester bond;         -   m) iteratively repeating steps j) through h) for each of the             consecutive nucleotide residues to be identified until the             final consecutive nucleotide residue is to be identified;         -   n) repeating steps j) and k) to identify the final             consecutive nucleotide residue, so as to thereby determine             the identity of each of the series of consecutive nucleotide             residues in the nucleic acid.

    -   In an embodiment in step c) the three types of dNTPs are chosen         from the group dATP, dCTP, dGTP and dTTP.

    -   In an embodiment the linker in each of step a) and f)         independently each comprise the structure:

or the structure:

wherein α represents a point of attachment to the base and β represents a point of attachment to the fluorophore, and wherein R is a cleavable chemical group.

-   -   In an embodiment a linker is cleaved by contacting the linker         with tris(2-carboxyethyl)phosphine.     -   In an embodiment one or more linkers are photocleavable or         chemically cleavable.     -   In an embodiment one or more chemical groups are photocleavable         or chemically cleavable.     -   In an embodiment R in the structures set forth in steps a) and         or f) is independently chosen from a —N₃ group or an allyl         group.     -   In an embodiment the cleavable chemical group in step f) is         independently chosen from a —N₃ group or an allyl group.     -   In an embodiment the first and second plurality of primers have         the same sequence.     -   In an embodiment one or more washing steps are performed in         between one or more of the steps set forth.

The methods described herein can be applied mutatis mutandis to sequencing RNA using the appropriate ddNTPS or analogues thereof and dNTPS and analogues thereof.

In the methods, base-pairing complementarity allows the sequence of the extended primer or of the target nucleic to be readily determined.

Dehybridize is understood by those skilled in the art to mean to disassociate the hybridized primer (or extended strand thereof) from the target nucleic acid without destroying the target nucleic acid and thus permitting further hybridization of a second primer to the target nucleic acid. Hybridization as used herein in one embodiment means stringent hybridization, for examples as described in Sambrook, J., Russell, D. W., (2000) Molecular Cloning: A Laboratory Manual: Third Edition.

Cold Spring Harbor Laboratory Press

“Type” of dNTP or ddNTP is used to distinguish dNTP or ddNTPs comprising different bases.

All combinations of the various elements described herein are within the scope of the invention.

This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details

Here, an alternative sequencing method that is a hybrid between Sanger dideoxy chain terminating reaction and sequencing by synthesis (SBS) is disclosed and some advantages that come with this hybrid sequencing approach are discussed. The fundamental difference between the two methods is that that the Sanger approach produces all possible complementary DNA extension fragments for a given DNA template and obtains the sequence after the separation of these fragments by reading the fluorescent labeled terminated base, while SBS relies on identification of each base as the DNA strand is synthesized by utilizing fluorescent labeled modified nucleotides that temporarily pauses the DNA synthesis for base identification. The limiting factors for increasing sequencing throughput in Sanger based method, as mentioned previously, is in the DNA separation using electrophoresis and limited parallelization of the capillaries. Concerns using SBS with labeled modified nucleotides lie on base modification where the labels are attached, and the fact that after cleavage of the label there may still remain a small trace of modification. This can affect the 3-D double helix structure of the DNA and, furthermore, the ability of DNA polymerase to bind to the double stranded DNA and efficiently incorporate the incoming nucleotide. The advantage in the Sanger based method is clearly in the Sanger dideoxy chain fragment producing reaction. It is a simple reaction, where DNA extensions are made with natural nucleotides. When the fluorescent labeled dideoxynucleotides are incorporated into a DNA strand, it no longer is involved in further DNA extension reactions. Therefore, the DNA polymerase extension reaction occurs with only natural substrates, and the efficiency of this reaction is clearly evident by its long read-length. The most attractive features in the SBS sequencing approach are the potentially massive parallel readout capability via high-density microarray and simplified sample preparation method. The integration of the advantageous features between the two methods to develop a hybrid DNA sequencing approach has been explored. The key molecular inventions suitable for the proposed hybrid DNA sequencing approach are rationally modified nucleotide and dideoxynucleotide analogue sets, which will allow generation of Sanger sequencing fragments on a DNA chip to produce sequencing data, thus bypassing the electrophoresis DNA separation steps.

Previous research efforts have firmly established the molecular level strategy to rationally modify the nucleotides by attaching a cleavable fluorophore to the base and capping the 3′-OH with a small capping moiety for SBS. Here, the design and synthesis of four photocleavable fluorescent dideoxynucleotides and four photocleavable nucleotide analogues as reversible terminators is disclosed for Sanger/SBS hybrid sequencing approach. Each of the four photocleavable dideoxynucleotide analogues contains a unique fluorophore with a distinct fluorescence emission at the base through a photocleavable linker. The four photocleavable reversible terminator nucleotides contain, in an embodiment, a 3′-O-(2-nitrobenzyl) group, which caps the 3′-OH on the sugar ring. It was first established that these dideoxynucleotide and nucleotide analogues are good substrates for DNA polymerase in a solution-phase DNA extension reaction and that the fluorophore and the 3′-O-(2-nitrobenzyl) group can be removed with high efficiency via laser irradiation at 355 nm in 10 sec. SBS was then performed using the combination mixture (such as in Sanger dideoxy chain termination reaction) of these 4 photocleavable fluorescent dideoxynucleotide analogues and 4 photocleavable 3′-O-modified reversible terminator nucleotides to accurately identify continuous bases of a DNA template immobilized on a chip. These results indicated that these photocleavable fluorescent dideoxynucleotides and photocleavable 3′-O-modified reversible terminator nucleotides can be rationally designed by attaching a cleavable fluorophore to the base of dideoxynucleotides and capping the 3′-OH with a small reversible moiety to the natural nucleotides so that they are still recognized by DNA polymerase as substrates. Furthermore, these analogues can generate 4-color DNA sequencing data by producing Sanger sequencing fragments on a sequencing by synthesis platform.

A modified inosine triphosphate (having a cleavable group attached to the 3′ O atom thereof) is used in the Sanger/sequencing by synthesis hybrid method to increase readlength and/or simplify the procedure. The dITP is used in conjunction with the ddNTPs, and as universal nucleotide can extend the nucleic acid by being complementary to any of the four natural nucleotides in the nucleic acid being sequenced.

Sequence by Synthesis with Template “Walking”

The fundamental rationale behind primer resetting is to regenerate the original primer site or to insert two or more primer sites of known sequences into the target DNA so SBS can be carried out at each site sequentially. In general, three steps are involved with this approach: 1) annealing of the first primer, 2) performing SBS, 3) denaturing the sequenced section of the template to recover a single-stranded DNA for the second primer annealing. These steps are carried out repeatedly until the target DNA is sequenced in its entirety. The advantage of primer resetting lies in its ability to restore all the templates after the denaturation step, including those that are terminated with ddNTPs, so the next cycle of SBS can restart with potentially the same amount of sequenceable DNA as the previous round.

Three approaches for achieving longer read lengths that rely on this template “walking” concept are described. In the first strategy, the DNA sequence is reset by reattaching the original primer, extending the chain with natural or minimally modified nucleotides to the end of the first round sequence, and then sequencing from that point. The second strategy relies on annealing of a second round primer that is longer than the first, containing at its 5′ end the same sequence as the original primer, followed by a run of 20 universal nucleotides such as inosine, from which the second round of sequencing can be primed. If the duplex stability of this highly degenerate primer with DNA templates is found to be low, a number of locked nucleotides can be added at either end of the primer to increase the stability of the primer-template complex. In the third strategy, extra priming sites are inserted within a template strand via Type IIS or Type III restriction-recircularization. Each of these approaches has distinct advantages and some difficulties that need to be overcome. None of the three aforementioned strategies are sensitive to the type of library (genomic, cDNA or other), to the method of amplification prior to sequencing (spotting of clones, ePCR, polony PCR), or the mode of sequencing (Hybrid SBS and SBS with C-F-NRTs). Hence they are all sequence unbiased, thus greatly increasing their range of applications in sequencing technologies.

Results

Design and Synthesis of Photocleavable Fluorescent Dideoxyribonucleotides for SBS.

To demonstrate the feasibility of carrying out de novo DNA sequencing by synthesis on a chip, four photocleavable fluorescent dideoxynucleotide analogues (ddCTP-PC-Bodipy-FL-510, ddUTP-PC-R6G, ddATP-PC-ROX and ddGTP-PC-Bodipy-650) (FIG. 1) were designed and synthesized as fluorescent terminators for DNA polymerase reaction. Modified DNA polymerases have been shown to be highly tolerant to nucleotide modifications with bulky groups at the 5-position of pyrimidines (C and U) and the 7-position of purines (A and G). Thus, each unique fluorophore was attached to the 5 position of C/U and the 7 position of A/G through a 2-nitrobenzyl linker. It was found that the fluorophore on a DNA extension product, which is generated by incorporation of the photocleavable fluorescent dideoxynucleotide analogues, are removed by 10 sec irradiation of the DNA extension product at 355 nm in aqueous solution. This rapid photocleavage reaction thus allows quantitative removal of the fluorophore after the DNA extension reaction. The detailed synthesis procedure and characterization of the 4 novel dideoxynucleotide analogues in FIG. 1 are described in Supporting materials and methods.

The efficient removal of the fluorophore from the DNA after detection is crucial for the SBS approach. Four photocleavable fluorescent dideoxynucleotides (FIG. 1) were synthesized and demonstrated that 10 seconds of irradiation with a laser at 355 nm led to the complete photocleavage of the fluorophore from the DNA. These nucleotide analogues were characterized by using them in a single base extension reaction and investigating the photocleavage of the fluorophore by UV irradiation. In order to verify that these fluorescent dideoxynucleotide analogues are incorporated accurately in a base-specific manner in a polymerase reaction, four separate DNA extensions and photocleavage via UV irradiation were carried out in solution. This allows the isolation of the DNA product at each step for detailed molecular structure characterization by MALDI-TOF mass spectrometry (MS) as shown in FIG. 2. The first extension product 5′-primer-A(PC-ROX)-3′ (1) was purified by HPLC and analyzed using MALDI-TOF MS [FIG. 2(A)]. This product was then irradiated at 355 nm using an Nd-YAG laser for 10 seconds and the photocleavage product was also analyzed using MALDI-TOF MS (FIG. 2(B)). The UV absorption by the aromatic 2-nitrobenzyl linker causes reduction of the 2-nitro group to a nitroso group and an oxygen insertion into the carbon-hydrogen bond located in the 2-position followed by cleavage and decarboxylation. As can be seen from FIG. 2(A), the MALDI-TOF MS spectrum consist of a distinct peak corresponding to the DNA extension product 5′-primer-A(PC-ROX)-3′ (m/z 9054), which confirms that the dideoxynucleotide analogue can be incorporated base specifically by DNA polymerase into a growing DNA strand. The small peak at m/z 8315 corresponding to the photocleavage product is seen due to the partial cleavage caused by the nitrogen laser pulse (337 nm) used in MALDI ionization. For the photocleavage experiment, near UV-irradiation at 355 nm by a laser was used for 10 seconds to cleave off the fluorophore from the DNA extension product. FIG. 2(B) shows the photocleavage result of the extension products. The extended mass peak at m/z 9054 completely disappeared while the peak corresponding to the photocleavage product 5′-primer-A appear as the sole dominant peak at m/z 8315, which establishes that laser irradiation completely cleaves the fluorophore with high speed and efficiency. DNA extension reactions using self-priming templates that incorporates the other three fluorescent dideoxynucleotide analogues as the first base were similarly carried out. As described above, the extension product 5′-primer-C(PC-Bodipy-FL-510)-3′ (FIG. 2(C)) was purified and analyzed by MALDI-TOF MS, then photocleaved (FIG. 2(D)) for further MS analysis. Extension products 5′-primer-G(PC-Bodipy-650)-3′ (FIG. 2(E)), 5′-primer-U(PC-R6G)-3′ (FIG. 2(G)) and their photocleavage to yield products 6 (FIG. 2(F)) and 8 (FIG. 2(H)) were similarly carried out and analyzed by MALDI-TOF MS. These results demonstrate that all four photocleavable fluorescent dideoxynucleotide analogues were correctly synthesized and successfully incorporated into the growing DNA strand in a polymerase reaction and the fluorophore can be efficiently cleaved by near UV irradiation, which makes it feasible to use these nucleotide analogues for SBS on a chip.

Design and Synthesis of 3′-Modified Photocleavable Nucleotides as Reversible Terminators for SBS.

A critical requirement for using SBS methods to sequence DNA unambiguously is a suitable chemical moiety to cap the 3′-OH of the nucleotide such that it terminates the polymerase reaction to allow the identification of the incorporated nucleotide. A stepwise separate addition of nucleotides with a free 3′-OH group has inherent difficulties in detecting sequences in homopolymeric regions. A 3′-OH capping group of the nucleotides allows for the addition of all four nucleotides simultaneously in performing SBS. This will decrease the number of cycles needed for sequencing with the requirement that the capping group then needs to be efficiently removed to regenerate the 3′-OH thereby allowing the polymerase reaction to proceed.

Four 3′-modified photocleavable nucleotides (3′-O-PC-dATP, 3′-O-PC-dCTP, 3′-O-PC-dGTP and 3′-O-PC-dTTP) as reversible terminators (FIG. 3) were synthesized and demonstrated that 10 seconds of irradiation with a laser at 355 nm led to the complete removal of the 3′-OH capping group (2-nitrobenzyl) from the DNA, thereby regenerating a free 3′-OH group for subsequent polymerase reaction. In order to verify that these fluorescent nucleotides incorporate accurately in a base specific manner, four continuous DNA extension reaction and photocleavage via UV irradiation were carried out in solution phase. This allowed the isolation of the DNA product at each step for detailed molecular structure characterization as shown in FIG. 4. The first extension product 5′-primer-T-3′-PC was purified by HPLC and analyzed using MALDI-TOF MS (FIG. 4(A)). This product was then irradiated at 355 nm using a laser for 10 seconds and the photocleavage product was also analyzed using MALDI-TOF MS (FIG. 4(B)). As can be seen from FIG. 4(A), the MALDI-TOF MS spectrum consist of a distinct peak corresponding to the DNA extension product 5′-primer-T-3′-PC (m/z 5965), which confirms that the nucleotide analogue can be incorporated base specifically by DNA polymerase into a growing DNA strand. The small peak at m/z 5830 corresponding to the photocleavage product is seen due to the partial cleavage caused by the nitrogen laser pulse (337 nm) used in MALDI ionization. FIG. 4(B) shows the photocleavage result on the extension product. The extended mass peak at m/z 5965 completely disappeared while the peak corresponding to the photocleavage product 5′-primer-T appear as the sole dominant peak at m/z 5830, which establishes that laser irradiation completely cleaves the 3′-OH capping group with high speed and efficiency. The next extension reaction was carried out using this photocleaved product, which now has a free 3′-OH group, as a primer to give a second extension product, 5′-primer-TG-3′-PC (FIG. 4(C)). As described above, the extension product was purified and analyzed by MALDI-TOF MS, then photocleaved (FIG. 4(D)) for further MS analysis. The third extension reaction to yield 5′-primer-UGA-3′-PC (FIG. 4(E)), the fourth extension to yield 5′-primer-UGAC-3′-PC (FIG. 4(G)) and their photocleavage to yield products 6 (FIG. 4(F)) and 8 (FIG. 4(H)) were similarly carried out and analyzed by MALDI-TOF MS. These results demonstrate that all four 3′-modified photocleavable nucleotide analogues were successfully incorporated into the growing DNA strand in a continuous polymerase reaction as reversible terminators and the 3′-OH capping group was efficiently cleaved by near UV irradiation.

4-Color DNA Sequencing by Synthesis on a Chip Using Photocleavable Fluorescent Dideoxynucleotide/3′-Modified Photocleavable Nucleotide Combination Remnant of Sanger Sequencing.

The combination of photocleavable fluorescent ddNTPs and 3′-modified photocleavable dNTPs were then used in an SBS reaction to identify the sequence of the DNA template immobilized on a solid surface. A site-specific 1,3-dipolar cycloaddition coupling chemistry was used to covalently immobilize the alkyne-labeled self-priming DNA template on the azido-functionalized surface in the presence of a Cu(I) catalyst. The principal advantage offered by the use of a self-priming moiety as compared to using separate primers and templates is that the covalent linkage of the primer to the template in the self-priming moiety prevents any possible dissociation of the primer from the template during the process of SBS. To prevent non-specific absorption of the unincorporated fluorescent nucleotides on the surface of the chip, a polyethylene glycol (PEG) linker is introduced between the DNA templates and the chip surface. This approach was shown to produce very low background fluorescence after cleavage to remove the fluorophore as demonstrated by the DNA sequencing data described below.

SBS was performed on a chip-immobilized DNA template using the photocleavable fluorescent ddNTPs and 3′-modified photocleavable dNTPs combination (ddCTP-PC-Bodipy-FL-510, ddUTP-PC-R6G, ddATP-PC-ROX and ddGTP-PC-Bodipy-650, 3′-O-PC-dATP, 3′-O-PC-dCTP, 3′-O-PC-dGTP and 3′-O-PC-dTTP) and the results are shown in FIG. 5. The structure of the self-priming DNA moiety is shown schematically in FIG. 5A, with the first 8 nucleotide sequences immediately after the priming site. The de novo sequencing reaction on the chip was initiated by extending the self-priming DNA using a solution containing the four 3′-O-PC-dNTPs as well as the four ddNTPs-PC-fluorophore, and 9° N mutant DNA polymerase. In order to negate any lagging fluorescence signal that is caused by previously unextended priming strand, a synchronization step was added to reduce the amount of unextended priming strands after the initial extension reaction. A synchronization reaction mixture consisting of just the four 3′-O-PC-dNTPs (FIG. 3) was used along with the 9° N mutant DNA polymerase to extend any remaining priming strand that has a free 3′-OH group to synchronize the incorporation. This extension method where the combination of 3′-O-PC-dNTPs/ddNTPs-PC-fluorophore are used, will not have a negative impact on the enzymatic incorporation of the next nucleotide analogue, because after cleavage to remove the 3′-OH capping group, the DNA product extended by 3′-O-PC-dNTPs carry no modification groups. Previous designs of cleavable fluorescent reversible terminators left small traces of modification (propargyl amine linker) after the cleavage of the fluorophore on the base of the nucleotide. Successive addition of these reversible terminators into a growing DNA strand during SBS will lead to a newly synthesized DNA strand with, at each base site, a small leftover linker. This may decrease the ability of the enzyme to efficiently incorporate the next incoming nucleotide, which will undoubtedly lead to loss of synchrony and furthermore, maximal readlength. With this combination approach, DNA products extended by ddNTPs-PC-fluorophore, after fluorescence detection and cleavage, are no longer involved in the subsequent polymerase reaction because they are permanent terminators. Therefore, further polymerase reaction only occurs on a DNA strand that incorporates the 3′-O-PC-dNTPs, and subsequently turned back into natural bases, which should have no ill effect on the enzyme. After washing, the extension of the primer by the complementary fluorescent dideoxynucleotide was confirmed by observing an orange signal (the emission from ROX) in a 4-color fluorescent scanner, [FIG. 5. (1)]. After detection of the fluorescent signal, the surface was immersed in dH₂O/acetonitrile (50/50, v/v) solution and irradiated at 355 nm for 1 min using a laser to cleave both the fluorophore from the DNA product extended with ddNTPs-PC-fluorophore and 3′-O-PC group from the DNA product extended with 3′-O-PC-dNTPs. The surface was then rinsed with dH₂O, and the residual fluorescent signal was detected again to confirm photocleavage, FIG. 5 (2). This was followed by another extension reaction using 3′-O-PC-dNTPs/ddNTPs-PC-fluorophore combination mixture to incorporate the next base complementary to the subsequent base on the template. The entire process of incorporation, synchronization, detection and cleavage was performed multiple times using the combination mixture of photocleavable fluorescent dideoxynucleotides and 3′-O-modified photocleavable reversible terminator nucleotides to identify 8 successive bases in the DNA template. The fluorescence image of the chip after each incorporation event is shown in FIG. 5B, while a plot of the fluorescence intensity vs. the progress of sequencing extension (raw 4-color sequencing data) is shown in FIG. 5C. The DNA sequences are unambiguously identified from the 4-color raw fluorescence data without any processing.

Strategy 1: Template “Walking” by Unlabeled Nucleotides

The fundamental rationale behind this template “walking” strategy is the removal of the sequenced strand and reattaching of the original primer to allow the extension, or walking, of the template with a combination of natural and modified nucleotides to the end of the first round sequence so that SBS can be carried out from that point. Since the original sequenced strand is stripped away, including those terminated with ddNTPs, all the templates become available for “walking”. Given that “walking” is carried out with either natural or 3′-modified nucleotides, the subsequent round of SBS is performed on nascent DNA strands for maximum read length. The advantage of template “walking” is its ability to restore all the templates after the denature step, includes those that are terminated with ddNTPs, so the next cycle of SBS can restart with potentially the same amount of nascent DNA as the previous round. The “walking” methodology is applicable to both hybrid SBS and SBS with C-F-NRTs, and has the potential to dramatically increase the read lengths of these SBS technologies (FIG. 9).

Template “Walking” for Hybrid SBS

1. Hybrid SBS (1^(st) Round)

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to efficiently decipher multiple DNA sequences in parallel. Hybrid SBS is a hybrid DNA sequencing method between the Sanger dideoxy chain terminating reaction and SBS. In this approach, four nucleotides (FIG. 10) modified as reversible terminators by capping the 3′-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates to extend the DNA chain, are used in combination with a small percentage of four cleavable fluorescent dideoxynucleotides (FIG. 11) to perform SBS. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs, while the role of the 3′-O-modified dNTPs is to further extend the DNA strand to continue the determination of the DNA sequence. Upon removing the 3′-OH capping group from the DNA products generated by incorporating the 3′-O-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination (FIG. 12). Such incorporation, fluorescence measurement and dye removal is repeatedly conducted until the detectable fluorescence intensity is not distinguishable, indicating a situation in which all the elongated primers are terminated with ddNTP. To overcome this “halted sequencing” due to ddNTP termination, a “walking” step is carried out to reset the templates.

2. Template “Walking”

Immediately after the first round of SBS, all of the elongated primers ended terminated with ddNTPs are removed from the template by denaturing. The templates are freed again and available for further sequencing reactions. To achieve template “walking”, the same starting primer is annealed to the template again and enzymatic incorporation is conducted to fill the gap between first and second stages of SBS. Five strategies are available for the walking process. Each approach has its advantages and shortcomings, which are summarized in the following.

Method 1.

-   -   Nucleotide reversible terminators (3′-O—R₁-dNTPs) are used as         substrates to perform enzymatic incorporation (FIG. 13). After         incorporation, specific chemical reaction is applied to         regenerate 3′-OH to ensure the subsequent incorporation. The         number of repeated cycles of such incorporation and cleavage         will exactly match the actual read length in the first stage of         SBS, so that this “filling gap” incorporation stops at the same         point where the longest ddNTP primer reaches.

Methods 2, 3, and 4.

-   -   Enzymatic incorporation is conducted using two sets of         nucleotides as substrates (FIG. 14). For example, the first set         of nucleotides composed of dCTP, dATP, and dGTP (sans dTTP) was         used to perform incorporation, so that the polymerase reaction         stops once it reaches a base “A” in template. Then enzymatic         incorporation is resumed with the second set of nucleotides         composed of dTTP, dATP, and dGTP (sans dCTP), resulting in a         polymerase reaction that stops at the base “G” in template. The         repeated cycles of such incorporations fill the gap between         first and second stages of SBS.     -   To minimize the mis-incorporation rate, another enzyme substrate         which can be recognized but not incorporated by the polymerase         is assigned to each set of nucleotides. For instance, if the DNA         polymerase used can only incorporate dNTP but not 3′ blocked         nucleotides, 3′-O—R₁-dTTP will be combined with dCTP, dATP and         dGTP as the first set, while 3′-O—R₁-dCTP will be combined with         dTTP, dATP and dGTP as the second set to elongate the primer         (FIG. 15).     -   Alternatively, deoxyribonucleotides diphosphate can also play         such role, replacing the 3′-O—R₁-dNTPs, during enzymatic         incorporation (FIG. 16).

Method 5.

-   -   Enzymatic incorporation is conducted using three dNTPs and         another nucleotide reversible terminator as substrates (FIG.         17). Primer elongation will only be stopped once it incorporates         nucleotide reversible terminator. After incorporation, specific         chemical reaction is applied to regenerate 3′-OH which ensure         consecutive incorporation of the next round. Repeated cycles of         such incorporation and cleavage will fill the gap between first         and second stages of SBS.         3. Re-Initiation of Hybrid SBS

Once the “walking” process is completed, the second stage of SBS is conducted using mixture of nucleotide reversible terminators and fluorescently labeled dideoxynucleotides as incorporation substrates same as described above. Another cluster of bases on the template can be continuously revealed, leading to the doubling of the original read length. The SBS-walking-SBS process is repeated to generate maximum read length.

EXAMPLES

1. Design and Synthesis of 3′-O-Modified NRTs and Cleavable Fluorescent Dideoxynucleotide Terminators for the Hybrid SBS

Four 3′-O-azidomethyl-modified NRTs (3′-O—N₃-dNTPs) were synthesized and evaluated (FIG. 18) for use in the hybrid SBS approach. The 3′-O-modified NRTs containing an azidomethyl group to cap the 3′-OH on the sugar ring were synthesized based on similar method to that reported by Zavgorodny et al. The 3′-O-azidomethyl group on the DNA extension product generated by incorporating each of the NRTs is efficiently removed by the Staudinger reaction using aqueous Tris(2-carboxy-ethyl) phosphine (TCEP) solution followed by hydrolysis to yield a free 3′-OH group for elongating the DNA chain in subsequent cycles of the hybrid SBS (FIG. 20A).

To demonstrate the feasibility of carrying out the hybrid SBS on a DNA chip, four cleavable fluorescent dideoxynucleotide terminators were designed and synthesized, ddNTP-N₃-Fluorophores (ddCTP-N₃-Bodipy-FL-510, ddUTP-N₃-R6G, ddATP-N₃-ROX and ddGTP-N₃-Cy5) (FIG. 19). The ddNTP-N₃-Fluorophore were used in combination with the four NRTs (FIG. 18) to perform the hybrid SBS. Modified DNA polymerases have been shown to be highly tolerant to nucleotide modifications with bulky groups at the 5-position of pyrimidines (C and U) and the 7-position of purines (A and G). Thus, a each unique fluorophore was attached to the 5 position of C/U and the 7 position of A/G through a cleavable linker. The cleavable linker is also based on an azido modified moiety as a trigger for cleavage, a mechanism that is similar to the removal of the 3′-O-azidomethyl group (FIG. 20B).

2. Four-Color DNA Sequencing on a Chip by the Hybrid SBS Approach

Hybrid SBS was performed on a chip-immobilized DNA template using the 3′-O—N₃-dNTP/ddNTP-N₃-fluorophore combination and the results are shown in FIG. 21.

The general four-color sequencing reaction scheme on a chip is shown in FIG. 21A. The de novo sequencing reaction on the chip was initiated by extending the self-priming DNA using a solution containing the combination of the four 3′-O—N₃-dNTPs and the four ddNTP-N₃-fluorophores, and 9° N DNA polymerase.

The four-color images from a fluorescence scanner for each step of the hybrid SBS on a chip is shown in FIG. 21B. The entire process of incorporation, synchronization, detection and cleavage was performed multiple times to identify 32 successive bases in the DNA template. The plot of the fluorescence intensity vs. the progress of sequencing extension (raw 4-color sequencing data) is shown in FIG. 21C. The DNA sequences were unambiguously identified with no errors from the 4-color raw fluorescence data without any processing.

3. Primer Reset and 2nd Round SBS

To demonstrate the concept of walking, the same self-priming DNA was immobilized on surface as template. After identifying the first 32 bases unambiguously with no errors by the first round hybrid SBS, the primer was reset for the second round SBS by elongating the original primer over the sequenced region via enzymatic incorporations. A solution containing dATP, dTTP, dCTP and 3′-O—N₃-dGTP was used to perform the polymerase reaction. 9° N DNA polymerase incorporates 3′ unblocked nucleotides more efficiently, leading to certain percentage of primers not fully extended by 3′-O—N₃-dGTP. To minimize this effect, a synchronization step was added to reduce the amount of out-of-phase primers after the initial extension reaction. A synchronization reaction mixture consisting of just 3′-O—N₃-dGTP in relative high concentration was used along with the 9° N DNA polymerase. The 3′-O-azidomethyl group on the DNA extension product generated by incorporating 3′-O—N₃-dGTP was efficiently removed by using aqueous Tris(2-carboxy-ethyl) phosphine (TCEP) solution to yield a free 3′-OH group for elongating the DNA chain in subsequent cycles of enzymatic incorporation. The entire process of incorporation, synchronization and cleavage were conducted repeatedly until the sequenced bases during the first round SBS were “walked” over. After the primer was reset by the enzymatic incorporation, the second stage of SBS was conducted using mixture of nucleotide reversible terminators and fluorescently labeled dideoxynucleotides as incorporation substrates same as described above. Another 13 bases were successfully identified after template “walking” (FIG. 22).

Template “Walking” for SBS with CFNRTs

1. SBS with C-F-NRTs

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to efficiently decipher multiple DNA sequences in parallel. Disclosed is the development of a DNA sequencing method that involves the extension of target DNA strand with modified cleavable fluorescent nucleotide reversible terminators (C-F-NRTs, 3′-O—R₁-dNTPs-R₂-fluorophore) in combination with cleavable nucleotide reversible terminators (C-NRTs, 3′-O—R₁-dNTPs). A set of four C-F-NRTs is produced via dual modifications by capping the 3′-OH group with a small chemical moiety and tethering a fluorophore through a cleavable linker to either the 7-position of the purines (A, G) or the 5-position of the pyrimidines (C, T) so that they are still recognized as substrates by DNA polymerase. Another set of four C-NRTs is modified similarly as the C-F-NRTs except no fluorophore is attached, which results in a reduction of the size of C-NRTs and the increment of DNA polymerase incorporation efficiency. In this approach, an extension mixture composed of the C-NRTs with a small percentage of the C-F-NRTs is used to perform SBS. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by the C-F-NRTs. Immediately following the detection step, a synchronization reaction is performed using only the C-NRTs to extend the un-extended DNA strands. A dideoxynucleotides (ddNTPs) capping step is carried out afterwards to completely rid of the remaining un-extended DNA. Upon removing the 3′-OH capping group from the DNA products generated by incorporating both C-F-NRTs and C-NRTs and the fluorophore from the C-F-NRTs, the polymerase reaction reinitiates to continue the sequence determination. The following scheme (FIG. 23) illustrates the general process for SBS with C-F-NRTs.

Four 3′-O—N₃-dNTPs-N₃-fluorophore (FIG. 24) and four 3′-O—N₃-dNTPs (FIG. 18) were synthesized, using an azidomethyl group as a chemically reversible capping moiety in the 3′-O-modified C-F-NRTs and C-NRTs, and an azido-based cleavable linker to attach the fluorophores to the C-F-NRTs,

After fluorescence detection for sequence determination, the azidomethyl capping moiety on the 3′-OH and the fluorophore attached to the DNA extension product via the azido-based cleavable linker are efficiently removed using tris(2-carboxyethyl)phosphine (TCEP) in aqueous solution compatible with DNA. Various DNA templates, including those with homopolymer regions were accurately sequenced with read length of over 20 bases using this SBS method on a chip and a four-color fluorescent scanner (FIG. 25).

Four C-F-NRTs (3′-O—N₃-dNTPs-N₃-fluorophore) were synthesize along with four C-NRTs (3′-O—N₃-dNTPs) for the implementation of our four-color de novo DNA sequencing by synthesis approach. During the incorporation stage of SBS, a mixture of the two sets of NRTs is used to extend the DNA strand. Only a small percentage of the 3′-O—N₃-dNTPs-N₃-fluorophore is used in the mixture so that the majority of the product is extended with the less bulky 3′-O—N₃-dNTPs. This approach leads to a more efficient DNA polymerase reaction since the smaller 3′-O—N₃-dNTPs are much easier to incorporate. Another advantage of having most of the DNA extended with 3′-O—N₃-dNTPs is the fact that after cleavage of the 3′-OH capping group on the product, nascent strand of DNA that have no traces of modification is restored. Such DNA does not have any adverse effect on the DNA polymerase during the subsequent incorporation of the complementary nucleotide. For DNA extended with the 3′-O—N₃-dNTPs-N₃-fluorophore, which serve as the signal producer, the 3′-OH is also restored after the cleavage step so that the next stage of SBS can be carried out. Therefore, it is possible to recover all the DNA templates after each round of sequencing, dramatically increasing the potential read-length of our SBS methodology. After the incorporation reaction, two separate capping steps, first with 3′-O—N₃-dNTPs and then with ddNTPs, are performed. The rationale behind the first capping reaction is to maximize the amount of extension products and to ensure the minimal loss of templates. In case there is any un-extended product after the first capping step, the second capping with ddNTPs is mostly likely to permanently terminate these DNA strands so that all templates are synchronized. Without these precautionary synchronization procedures, mixed fluorescent signals will prevent the identification of the correct nucleotide incorporated. Since both 3′-O—N₃-dNTPs-N₃-fluorophore and 3′-O—N₃-dNTPs are reversible terminators, which allow the sequencing of each base in a serial manner, they can accurately determine the homopolymeric regions of DNA. In addition, due to the fact that all of the steps of our SBS approach are performed on a DNA chip, there is no longer a need for electrophoretic DNA fragment separation as in the classical Sanger sequencing method.

Even though theoretically SBS with C-F-NRTs can be executed without losing templates, the utilization of ddNTPs capping does reduce the number of available templates during the actual sequencing reaction. In addition, the incorporation and cleavage of C-F-NRTs leave a tail on the modified nucleotides that can potentially reduce the incorporation efficiency the subsequent base. Hence template “walking” can be applied to increase read length for this SBS methodology.

2. Template “Walking”

Immediately after the first round of SBS, DNA templates are denatured by heat or mild alkali conditions to rid of the extended primer. The same original primer is re-hybridized to the template chain, and one of the five “walking” methods described in the previous section can be applied to reset the start point for the next round of SBS at the end of the first sequencing run (FIGS. 26, 27, 28, 29, and 30).

3. Re-Initiation of SBS with C-F-NRTs

Once the “walking” process is completed, the primer is extended to the end of the previous round of SBS. At this point, hybrid SBS is carried out to identify the subsequent bases. If the process can be repeated more times, it should be theoretically possible to achieve long and significant read length.

Strategy 2: Template “Walking” with Universal Bases

In this variation on the Strategy 1, the reset is achieved not with nucleotide walking, but with the use of a longer primer partially consisting of universal nucleotides for the second round. Attachment of the template DNA to the surface and the first few steps of the procedure are identical to the first method. However, after stripping the first extended primer for the initial 20 base readout, a long primer with the following features will be hybridized to the template: (a) the first half is identical to the initial primer; (b) the second half is composed almost entirely of universal bases. One possible candidate for the universal base is inosine, which, in its deoxynucleoside form, can base pair with all four nucleotides, though its affinity for C and A is significantly higher than for G and T; a second possibility is 5-nitroindole; (c) the last one or two anchoring bases of the long primers are degenerate with each of the four possible bases being represented. Because the universal bases can form hydrogen bonds with any of the other four bases with some efficiency, they have the capacity to bind to the first 20 or so bases of the sequence. However, the melting temperature of the ensuing hybridization is reduced substantially by the run of inosines, a few of the bases in the first half and the two 3′-anchoring bases can be substituted with locked nucleotides. Locked nucleic acids have a chemical bond between the 2′ and 4′ carbons of the ribose. While slower to associate with their complementary base, once hybridized, they tend not to dissociate. Thus, they provide a nice solution to ensure that the long primer remains attached appropriately to the template. In addition, the percentage of locked nucleosides in the primer can be manipulated to achieve higher hybridization strength. After hybridization of the above long primer, a second round of either Hybrid SBS or SBS with C-F-NRTs can be performed (FIG. 31).

An alternative approach to Strategy 2 is the use of a detachable loop primer, possibly with a labile sugar and glycosylase treatment. After the first round of sequencing, the loop is removed by enzymatic cleavage and denaturation, and then a new identical loop is attached. In a modification that is a composite of “walking” Strategy 1 and 2, the new loop primer can be composed of an initial portion identical to the first loop primer, a “loop out” region that bypasses the first set of sequenced nucleotides, and a degenerate anchoring nucleotide to initiate the second round of sequencing.

Strategy 3: Multiple Primers “Walking”

In this third strategy, one or two additional primer annealing sites are introduced into the DNA to be sequenced at a distance just about equal to the number of bases that can be sequenced from the first primer.

As illustrated in FIG. 32, template preparation for SBS will utilize the cloning of genomic DNA into a specially designed vector containing type IIS or III restriction sites (MmeI and EcoP15 I) flanking the genomic DNA cloning site. In this procedure size fractionated DNA (minimal length 100 bp) will be ligated into the cloning vector using blunt-end ligation. Upon cloning, the resulting recombinant plasmids will be re-cut at one of the type IIS/III sites and the sticky ends will be filled in with Klenow enzyme. Next, specific sequencing primers will be introduced via ligation inside the genomic DNA inserts, 22 bases distant from the first primer in the case of Nine′ or 27 bases away in the case of EcoP15 I. After insertion of the internal priming sites, the constructs will be re-cloned in E. coli, the recombinant plasmids isolated and the inserts re-amplified by PCR at vector-insert junctions and attached to the beads for sequencing. Alternatively, emulsion or polony PCR strategies can be used to accomplish attachment of single molecules to individual beads or slide locations and their subsequent amplification at a much lower cost than cloning. In any case, once the DNA is immobilized, the first round of Hybrid SBS or SBS with C-F-NRTs will be primed from the flanking primer, then after stripping these extended primers, the second set of sequencing reactions will be initiated at the internal primer. It should be noted that with this scheme, the two sequenced portions come from opposite ends of the initial DNA, and are in essence paired end reads.

Several novel modifications of this approach can address the desire of many investigators to sequence an entire 100-base stretch of DNA, the length of a typical exon including surrounding intronic bases adjacent to the splice site. For instance, one can prepare a construct with two internal primers. In this case, the initial vector will be designed with MmeI at one flank and EcoP15I on the other; using two consecutive restriction, cloning and circularization steps, the final construct will consist of four alternative priming sites (two on the insert flanks and two internal), which in the case of 100 bp segments of genomic DNA will guarantee their complete sequencing with 25-30 cycles of SBS and three primer resets. The extra cycles would enable some of the sequence reads to run into the next primer, which would help to confirm the direction (e.g., the last sequence might end with the MmeI or EcoP15I site. Other tricks would include modifying the ends of the primers to allow looping and reverse direction sequencing, incorporation of one or two decoding bases in the internal primers to confirm directions, and deconvoluting the results after all the data is generated. One would want to have a single set of primers for sequencing, regardless of which strand is attached. In order to achieve this, and to overcome the non-directional nature of their insertion, the internal primer or primers will be designed as palindromes so that sequencing can be initiated in either direction.

Materials and Methods

DNA Polymerase Reaction Using Four Photocleavable Fluorescent Dideoxynucleotides.

It was previously demonstrated that a library of nucleotide analogue, dNTPs-PC-fluorophore, can be efficiently incorporated by DNA polymerase in a DNA extension reaction and the fluorophore can be effectively cleaved off by laser irradiation at 355 nm (1-4). Here, four dideoxynucleotide analogues have been characterized, ddCTP-PC-Bodipy-FL-510, ddUTP-PC-R6G, ddATP-PC-ROX and ddGTP-PC-Bodipy-650 that were used for SBS on a chip by performing four separate DNA-extension reactions, each with a different template allowing the four ddNTP analogues to be incorporated. The self-priming template (a 26-mer hairpin DNA with a 4-base 5′-overhang) sequences were (5′-GACTGCGCCGCGCCTTGGCGCGGCGC-3′ (SEQ ID No:1)) for ddATP-PC-ROX incorporation, (5′-ATCGGCGCCGCGCCTTGGCGCGGCGC-3′ (SEQ ID No:2)) for ddCTP-Bodipy-FL-510 incorporation, (5′-GATCGCGCCGCGCCTTGGCGCGGCGC-3′ (SEQ ID No:3)) for ddGTP-PC-Bodipy-650 incorporation and (5′-GTCAGCGCCGCGCCTTGGCGCGGCGC-3′ (SEQ ID No:4)) for ddUTP-PC-R6G incorporation. Each of the extension reactions consisted of using a mixture of all four ddNTPs-PC-fluorophore (120 pmol of ddCTP-PC-Bodipy-FL-510, 120 pmol of ddUTP-PC-R6G, 120 pmol of ddATP-PC-ROX and 120 pmol of ddGTP-PC-Bodipy-650) along with 60 pmol of the self-priming template, 2 μL of 10× Thermopol reaction buffer (New England Biolabs), and 2 units of 9° N mutant DNA polymerase (exo-) A485L/Y409V in a total reaction volume of 20 μL. The reaction consisted of incubations at 94° C. for 5 min, 4° C. for 5 min, and 62° C. for 20 min. Subsequently, the extension product was purified by using a reverse-phase HPLC. An Xterra MS C18 (4.6×50-mm) column (Waters) was used for the HPLC analysis. Elution was performed over 120 minutes at a flow rate of 0.5 mL/min with the temperature set at 50° C. by using a linear gradient (12-34.5%) of methanol in a buffer consisting of 8.6 mM triethylamine and 100 mM hexafluoroisopropyl alcohol (pH 8.1). The fraction containing the desired product was collected and freeze-dried for mass spectrometry analysis using Voyager DETM MALDI-TOF mass spectrometer (Applied Biosystems) and photocleavage. For photocleavage, the purified DNA extension product bearing the deoxynucleotide analogue was resuspended in 200 μL of deionized water. The mixture was irradiated using a laser for 10 seconds at 355 nm and then analyzed by MALDI-TOF MS. After photocleavage, the DNA fragment with the fluorophore removed was used as a primer for a second extension reaction using dGTP-PC-Bodipy-FL-510 (reaction mixture described above). The second extended product was then purified by HPLC as described previously and photolyzed. The third extension using dATP-PC-ROX and the fourth extension using dCTP-PC-Bodipy-650 were carried out in a same manner using the previously extended and photocleaved product as their primer.

Continuous DNA Polymerase Reaction Using Four 3′-O-Modified Photocleavable Nucleotides as Reversible Terminators in Solution.

The four nucleotide analogues 3′-O-PC-dATP, 3′-O-PC-dCTP, 3′-O-PC-dGTP and 3′-O-PC-dTTP have been characterized, by performing four continuous DNA-extension reactions sequentially using a primer (5′-AGAGGATCCAACCGAGAC-3′ (SEQ ID No:5)) and a synthetic 60-mer DNA template (5′-GTGTACATCAACATCACCTACCACCATGTCA-GTCTCGGTTGGATCCTCTATTGTGTCCGG-3′ (SEQ ID No:6)) based on a portion of exon 7 of the human p53 gene. The four nucleotides in the template immediately adjacent to the annealing site of the primer are 3′-ACTG-5′ (SEQ ID No:7). First, a polymerase extension reaction using a pool of all four nucleotide analogues along with the primer and the template was performed producing a single base extension product. The reaction mixture for this, and all subsequent extension reactions, consisted of 80 pmol of template, 60 pmol of primer, 120 pmol of 3′-O-PC-dNTPs, 1× Thermopol II reaction buffer, 40 nmol of Mn2+ and 2 units of 9° N mutant DNA polymerase (exo-) A485L/Y409V in a total reaction volume of 20 μL. The reaction consisted of 20 cycles at 94° C. for 20 sec, 48° C. for 40 sec, and 62° C. for 90 sec. Subsequently, the extension product was purified by using reverse-phase HPLC. The fraction containing the desired DNA product was collected and freeze-dried for analysis using MALDI-TOF mass spectrometry. For photocleavage, the purified DNA extension product bearing the 3′-O-modified nucleotide analogue was resuspended in 200 μL of deionized water. The mixture was then irradiated using a laser for 10 seconds at 355 nm and characterized by MALDI-TOF MS. The DNA product with the 3′-O-(2-nitrobenzyl) group removed to generate a free 3′-OH group was used as a primer for a second extension reaction using 3′-O-PC-dNTPs. The second extended DNA product was then purified by HPLC and photocleaved. The third and the fourth extensions were carried out in a similar manner using the previously extended and photocleaved product as the primer.

4-Color DNA Sequencing by Synthesis on a Chip Using Photocleavable Fluorescent Dideoxynucleotide/3′-Modified Photocleavable Nucleotide Combination Remnant of Sanger Sequencing.

Ten microliters of a solution consisting of ddCTP-PC-Bodipy-FL-510 (25 fmol), ddUTP-PC-R6G (50 fmol), ddATP-PC-ROX (100 fmol), ddGTP-PC-Bodipy-650 (100 fmol), 3′-O-PC-dATP (14 pmol), 3′-O-PC-dCTP (3.5 pmol), 3′-O-PC-dGTP (14 pmol), 3′-O-PC-dTTP (7 pmol) [a 3′-O-PC-dNTPs:ddNTPs-PC-fluorophore ratio of 140:1], 1 U of 9° N mutant DNA polymerase, and 1× reaction buffer was spotted on the surface of the chip, where the self-primed DNA moiety was immobilized. The base complementary to the DNA template was allowed to incorporate into the primer at 62° C. for 10 min. To synchronize any unincorporated templates, an extension solution consisting of 30 pmol each of 3′-O-PC-dCTP, 3′-O-PC-dTTP, 3′-O-PC-dATP and 3′-O-PC-dGTP, 1 U of 9° N mutant DNA polymerase, and 1× reaction buffer was spotted on the same spot and incubated at 62° C. for 10 min. After washing the chip with a SPSC buffer containing 0.1% Tween 20 for 1 min, the surface was rinsed with dH₂O, dried briefly and then scanned with a 4-color ScanArray Express scanner (Perkin-Elmer Life Sciences) to detect the fluorescence signal. The 4-color scanner is equipped with four lasers with excitation wavelengths of 488, 543, 594, and 633 nm and emission filters centered at 522, 570, 614, and 670 nm. To perform the photocleavage, the glass chip was placed inside a chamber filled with dH₂O/acetonitrile (1/1 v/v) solution and the spot where the self-primed DNA moiety is immobilized was irradiated for 1 min with a laser at 355 nm. After washing the surface with dH₂O, the chip was scanned again to compare the intensity of fluorescence after photocleavage with the original fluorescence intensity. This process was followed by the next polymerase extension reaction using the ddNTPs-PC-fluorophore/3′-O-PC-dNTPs combination mixture (ratio adjusted to 120:1, with concentration of ddNTPs-PC-fluorophore remaining the same), with the subsequent synchronization, washing, fluorescence detection, and photocleavage processes performed as described above. With each subsequent polymerase extension reaction, the ratio was adjusted, decreasing in increments of 20 (100:1 to 80:1 to 60:1 and so on), until the mixture consisted only of ddNTPs-PC-fluorophore. This SBS cycle was repeated multiple times using the combination mixture of ddNTPs-PC-fluorophore/3′-O-PC-dNTPs in each polymerase extension reaction to obtain de novo DNA sequencing data on a DNA template immobilized on a chip.

Discussion

Four photocleavable fluorescent dideoxynucleotide analogues have been synthesized and characterized along with four 3′-O-modified photocleavable reversible terminator nucleotides and used them to produce 4-color de novo DNA sequencing data on a chip by Sanger/SBS hybrid sequencing approach. In doing so, the combining of the advantageous aspects of Sanger and SBS sequencing approaches to sequence DNA unambiguously was achieved. First, a strategy to use a photocleavable reversible moiety to cap the 3′-OH group of the nucleotide has been successfully implemented so that the nucleotide temporarily terminates the polymerase reaction to allow the identification of the incorporated photocleavable fluorescent dideoxynucleotide. After photolysis, both the fluorophore from the dideoxynucleotide and the 2-nitrobenzyl group from the 3′-O-PC modified reversible terminator are removed. Removal of the fluorophore after the identification of the base is crucial so that it does not interfere with the fluorescence detection of the next incorporated base. Regeneration of the 3′-OH group is needed for the subsequent incorporation of the next complementary base. With these 3′-O-modified reversible terminators, after photocleavage, there are no traces of modification, thereby regenerating a natural nucleotide at the terminal 3′-end of growing DNA strand. Furthermore, photocleavage reaction is catalyzed by photons and introduces no additional chemical reagents to cleave the fluorophore and the 3′-O-(2-nitrobenzyl) group, which eliminates possible chemical residue contamination during the subsequent polymerase reaction. Therefore, there will be no adverse effect on the DNA polymerase for the incorporation of the next complementary base. Second, both these modified dideoxynucleotide and nucleotide analogues are terminators (permanent and reversible) which allow the interrogation of each base in a serial manner, a key procedure enabling accurate determination of homopolymeric regions of DNA template. Because the fluorescence-driven base identification is made as each complementary base is incorporated and subsequently cleaved off, it enables the elimination of DNA fragment separation procedure. It also allows for the addition of all 8 substrates simultaneously in performing Sanger/SBS hybrid sequencing. This ultimately reduces the number of cycles needed to complete the sequencing cycle, increases sequencing accuracy due to competition among the substrates in the polymerase reaction.

The key factors governing the sequencing readlength of our 4-color Sanger/SBS hybrid sequencing approach are: 1) the purity of 3′-O-modified photocleavable reversible terminators. Any impurities even in small amounts, such as nucleotides with a free 3′-OH, where the capping group has come off, will significantly affect the synchrony of the DNA extension; 2) the ratio of 3′-O-PC-dNTPs to ddNTPs-PC-fluorophore. Because the amount of self-priming DNA template is set before the first incorporation, during each polymerase reaction, most of the DNA self-priming DNA template should be extended with 3′-O-modified reversible terminators and only fractional amount extended with fluorescent dideoxynucleotides. Once the dideoxynucleotides incorporate into the growing DNA strand, that strand no longer will participate in further DNA synthesis. One way to achieve this is to have a large 3′-O-PC-dNTPs to ddNTPs-PC-fluorophore ratio so that it will lead to prolonged DNA extension.

Due to the high detection sensitivity of the scanner, small amount of fluorophore is sufficient to detect its fluorescence emission; 3) the yield of cleavage of the fluorophore and the 3′-OH capping group from the DNA extension products. The photocleavage has near quantitative yield in solution phase experiments. The yield on the surface is difficult to measure precisely due to the small non-specific absorption of fluorophores affecting the background fluorescence level. The strong fluorescence signal to background ratio (˜20:1) seen in FIG. 5 indicates that the readlength should be able to be extended even further. A signal to background ratio of 3 or 4 to 1 should be sufficient to accurately determine the DNA sequence. In terms of readlength, Sanger sequencing holds the standard with readlength of over 700 bp, which is encouraging for this hybrid sequencing approach. With the massive parallel output capability, this hybrid approach may potentially achieve the necessary throughput and cost reduction that defines the next-generation sequencing platform. The basic principle and strategy outlined in the 4-color Sanger/SBS hybrid approach will be built on by improving in the areas of DNA extension methodology with engineering of high performance polymerases tailored for both the photocleavable fluorescent dideoxynucleotides and 3′-O-modified reversible terminators, as well as investigating into alternative cleavable linkers and 3′-OH reversible capping moieties.

REFERENCES

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What is claimed is:
 1. A method for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid template comprising: a) contacting a plurality of the nucleic acids with (i) at least four different dideoxynucleotide triphosphate (ddNTP) analogues, each having the structure:

wherein F is a fluorophore, Base is a base which is adenine, guanine, cytosine, uracil or thymine, wherein the fluorophore attached through a linker to each type of base differs in its emission or excitation spectra from a fluorophore attached to each of the remaining types of bases, and each of the four ddNTP analogues differs from the remaining three ddNTP analogues by having a different base, wherein L is a cleavable linker molecule,  (ii) at least four deoxynucleotide triphosphate (dNTP) analogues having the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil, or thymine, and wherein R′ is a cleavable chemical group, wherein each of the four dNTP analogues differs from the remaining three dNTP analogues by having a different base,  (iii) a nucleic acid polymerase and  (iv) a plurality of a first nucleic acid primer which can each hybridize with a separate one of each of the plurality of nucleic acids,  under conditions permitting (A) one of the four ddNTP analogues that is complementary to the consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of one of the first nucleic acid primers and thereby extend that primer and (B) one of the four dNTP analogues that is complementary to a consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of another one of the first nucleic acid primers and thereby extend that primer; b) identifying the fluorophore of the ddNTP analogue which has formed the phosphodiester bond, thereby identifying the consecutive nucleotide; c) cleaving the linker attaching the fluorophore of the ddNTP analogue which has formed the phosphodiester bond and cleaving the cleavable chemical group from the dNTP which has formed the phosphodiester bond; d) iteratively repeating steps a) through c) for each of the consecutive nucleotide residues to be identified until the final consecutive nucleotide residue is to be identified; e) repeating steps a) and b) to identify the final consecutive nucleotide residue, f) denaturing the extended first nucleic acid primers so that they de-hybridize them from the plurality of nucleic acid templates; g) contacting the plurality of nucleic acids with (i) three different types of deoxynucleotide triphosphates (dNTPs), (ii) a nucleic acid polymerase and (iii) a first substrate of the polymerase, wherein the substrate can be recognized but not incorporated into the growing DNA strand by the polymerase, wherein the substrate is complementary to a nucleotide residue that is different from each of the nucleotide residues that the three dNTPs are complementary to, respectively, and (iv) a plurality of a second nucleic acid primer which can each hybridize with a separate one of the plurality of nucleic acid templates, under conditions permitting one of the three dNTP that is complementary to the consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of one of the second nucleic acid primers and thereby extend that second primer; h) contacting the plurality of the nucleic acid template with (i) three different types of deoxynucleotide triphosphates, wherein at least one type of the deoxynucleotide triphosphates is not used in step g), and (ii) a second substrate of the polymerase, wherein the substrate can be recognized but not incorporated to the growing DNA strand by the polymerase, wherein the substrate is complementary to a nucleotide residue that is different from each of the nucleotide residues that the three dNTPs are complementary to, respectively, under conditions permitting one of the three dNTPs that is complementary to the consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of one of the plurality of the extended second nucleic acid primer and thereby extend that primer; i) iteratively repeating steps g) and h) until the second nucleic acid primer is extended up to and including a residue corresponding to the final consecutive nucleotide residue identified in step e); j) contacting the plurality of extended second nucleic acid primers with (i) at least four different dideoxynucleotide triphosphate (ddNTP) analogues, each having the structure:

wherein F is a fluorophore, Base is a base which is adenine, guanine, cytosine, uracil or thymine, wherein the fluorophore attached through a linker to each type of base differs in its emission or excitation spectra from a fluorophore attached to each of the remaining types of bases, and each of the four ddNTP analogues differs from the remaining three ddNTP analogues by having a different base, wherein L is a cleavable linker molecule,  (ii) at least four deoxynucleotide triphosphate (dNTP) analogues having the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil, or thymine, and wherein R′ is a cleavable chemical group, wherein each of the four dNTP analogues differs from the remaining three dNTP analogues by having a different base, and  (iii) a nucleic acid polymerase,  under conditions permitting (A) one of the four ddNTP analogues that is complementary to the next consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of one of the extended second nucleic acid primers and thereby extend that second nucleic acid primer and (B) one of the four dNTP analogues that is complementary to a consecutive nucleotide residue to be identified to form a phosphodiester bond with the 3′ end of another one of the extended second nucleic acid primers and thereby extend that second primer; k) identifying the fluorophore of the ddNTP analogue which has formed the phosphodiester bond, thereby identifying the consecutive nucleotide; l) cleaving the linker attaching the fluorophore of the ddNTP analogue which has formed the phosphodiester bond and cleaving the cleavable chemical group from the dNTP which has formed the phosphodiester bond; m) iteratively repeating steps j) through l) for each of the consecutive nucleotide residues to be identified until the final consecutive nucleotide residue is to be identified; n) repeating steps j) and k) to identify the final consecutive nucleotide residue,  so as to thereby determine the identity of each of the series of consecutive nucleotide residues in the nucleic acid.
 2. The method of claim 1, wherein the linker in each of step a) and j) independently each comprise the structure:

or the structure:

wherein α represents a point of attachment to the base and β represents a point of attachment to the fluorophore.
 3. The method of claim 1, wherein a linker is cleaved by contacting the linker with tris(2-carboxyethyl)phosphine.
 4. The method of claim 1, wherein one or more linkers are photocleavable or chemically cleavable.
 5. The method of claim 1, wherein one or more chemical groups are photocleavable or chemically cleavable.
 6. The method of claim 1, wherein R′ in the structures set forth in steps a) and or j) is independently chosen from a —N₃ group or an allyl group.
 7. The method of claim 1, wherein the first and second plurality of primers have the same sequence.
 8. The method of claim 1, wherein one or more washing steps are performed in between one or more of the steps set forth.
 9. The method of claim 1, wherein in step g) the three types of dNTPs are selected from the group consisting of dATP, dCTP, dGTP and dTTP.
 10. The method of claim 1, wherein in step g) the substrate is a deoxynucleotide diphosphate (dNDP).
 11. The method of claim 1, wherein in step g) the substrate is a deoxynucleotide triphosphate analogue having a blocking group at the 3′-O atom.
 12. The method of claim 1, wherein the plurality of nucleic acids are contacted with the four different ddNTP analogues and four different dNTP analogues in a ddNTP analogues:dNTP analogues ratio of about 1:10, 1:50, 1:250, or 1:500. 